Recombinant TNF ligand family member polypeptides with antibody binding domain and uses thereof

ABSTRACT

The present invention relates in general to the field of TNF ligand family members. In more detail the present invention relates to polypeptides comprising at least three components A, each of which comprises the sequence of a TNF homology domain (THD) of a TNF ligand family member, or a functional derivative thereof, and comprising at least one component B consisting of a VL region and a VH region linked directly to each other with a linker sequence L which has a length of &lt;12 amino acids. Furthermore, the present invention also relates to nucleic acids encoding such polypeptides and pharmaceutical compositions thereof.

This application is a continuation of U.S. application Ser. No.14/009,186, filed Aug. 22, 2014, which is a U.S. National StageApplication of International Application No. PCT/EP2012/001426, filedMar. 30, 2012, and claims the benefit of European Patent Application No.EP 11002745.5, filed Apr. 1, 2011, the disclosures of each of which areexplicitly incorporated by reference herein in their entirety.

The present invention relates in general to the field of TNF ligandfamily members. In more detail the present invention relates topolypeptides comprising at least three components A, each of whichcomprises the sequence of a TNF homology domain (THD) of a TNF ligandfamily member, or a functional derivative thereof, and comprising atleast one component B consisting of a V_(L) region and a V_(H) regionlinked directly to each other with a linker sequence L which has alength of ≤12 amino acid residues. Furthermore, the present inventionalso relates to nucleic acids encoding such polypeptides.

The members of the TNF ligand family are proinflammatory cytokines.Cytokines in general, and in particular the members of the TNF ligandfamily, play a crucial role in the stimulation and coordination of theinnate immune system as well as in the adaptive immune response(including both cellular and humoral immunity), induction of apoptosis,bone architecture, hair growth, teeth growth, development ofperspiratory glands, lymph node development and many more (Aggarwal, B.B. (2003), Nat. Rev. Immunol. 3, 745-756). A dysregulation of theactivity of members of the TNF ligand family can, however, lead to amultitude of pathologies. This includes for example septic shock,autoimmune diseases like rheumatoid arthritis, or neurodegenerativediseases. Tumor necrosis factor (TNF) is the name-giving and probablymost important member of this large cytokine family.

Members of the TNF ligand family exert their biological function ashomotrimers (Banner, D. W. et al, (1993), Cell 73, 431-445). Trimericstructures and likewise aggregations of higher order (e.g. oligomers ormultimers of trimers) of such proteins may be encountered frequently innature. Examples for this are the Cartilage Matrix Protein (CMP), whichis a connective tissue protein (Beck et al. (1996), J. Mol. Biol. 256,909-923), proteins of the family of collagens, like the Clq family towhich Clq, collagen α1 (X), α2 (VII), the hibernation protein, ACRP30,the inner ear structure protein, cellebrin and multimerin belong(Kishore and Reid (1999), Immunopharmacol. 42, 15-21), and proteins ofthe collectin family such as lung surfactant protein A (SP-A) andmannose binding protein (MBP) (Epstein et al., (1996), Current Opinionin Immunology, Vol. 8, No. 1, 29-35).

Trimer formation occurs due to interactions between the individualmonomers such as hydrophobic interactions, hydrogen bridges, covalentbonds (e.g. disulfide bonds), and/or Coulomb forces, but also occurs onbasis of structural motifs, i.e. specific amino acid sequences whichlead to formation of intermolecular supersecondary structures. In caseof the members of the TNF ligand family, the three monomers associate inthe homotrimeric structure via non-covalent hydrophobic bonds. In theactive form they in turn activate members of the TNF receptor familywhich themselves do not possess any enzymatic activity. For example, TNFbeing a member of the TNF ligand family binds to the two membranereceptors TNFR1 and TNFR2 and mediates the oligomerization and theactivation of inactive receptors. As a consequence of the receptorcomplex formation a signal cascade is initiated which brings about interalia an association of cytoplasmatic adaptor proteins (Wajant, H. et al.(2003), Cell Death, Differ, 10, 45-65). The interaction of TNFR1 andTNFR2 with its ligand is characterized by binding of the receptors atthe space between two of the three TNF monomers of the TNF homotrimer(Banner (1993), supra). This illustrates that TNF as well as all theother members of the TNF ligand family are only biologically active inthe homotrimeric structure. Due to their function, members of the TNFligand family and the respective membrane receptors, respectively, maybe used for many kinds of treatments of diseases, such as infectiousdiseases, inflammatory diseases, metabolic diseases, diseases resultingfrom a dysregulation of apoptosis, neurodegenerative diseases and manymore diseases. They play a particularly important role in the treatmentof cancerous diseases because members of the TNF ligand family exhibitin general an antitumor activity. This applies in particular to TNF(Eggermont, A. M. and ten Hagen, T. L. (2003), Curr. Oncol. Rep. 5,79-80), TRAIL (TNF related apoptosis using ligand), also termed Apo 2L(Weley et al. (1995), Immunity 6: 673-682; Petti et al. (1996), J. Biol.Chem. 271: 12687-12689) and FasL (CD95L). However, in vivo experimentsshowed strong systematic adverse reactions for TNF and agonists of theFas receptor, and in vitro experiments indicate similarly toxic effectsfor certain TRAIL compounds (Jo et al. (2000) Nat Med 6: 564-567,Ichikawa et al. (2001) Nat Med 7: 954-960; Ogasawara et al. (1993)Nature 364: 806-809). For this reason, the clinical use of Fasactivating ligands/agonists in a systemic application has so far beenconsidered impossible due to safety concerns. However, due to theimportance of TNF, TRAIL, FasL (CD95L) and other TNF ligand familymembers for this field, and due to the adverse reactions associated withtheir application in form of a clinical systemic administration, severalalternative approaches were pursued in order to minimize the adversereactions (Eggermont, A. M. and ten Hagen, T. L. (2003), Curr. Oncol.Rep. 5, 79-80).

WO 02/22680 describes for example fusion proteins which allow a targetedand tissue and/or cell-specific activity of cytokines by fusing thecytokine with an antigen-binding antibody. By this means it is achievedthat the cytokine is not active on tissue or cells, respectively, whichare not in contact with these fusion proteins, and that adversereactions regarding these tissues and cells, respectively, are reduced.

In DE 102 47 755 an antibody-independent system is disclosed, whichlikewise allows for a targeted, tissue- and cell-specific action of thecytokines. The document discloses fusion proteins with a biologicallyactive domain and a cell surface binding domain. Biological activity ofthe biologically active domain is mediated by binding of the cellsurface binding domain to the respective cell surface. Besides ofproviding a reduction of adverse reactions for non-target tissue, thissystem may also be advantageously used for cell surface molecules ontarget cells for which no or only low specificity antibodies areavailable.

Apart from the above-mentioned adverse reactions it is additionallyproblematic that the active homotrimers of members of the TNF ligandfamily dissociate even at physiologically reasonable concentrations.This dissociation is reversible; however, the protein quickly loses itsbioactivity because it is denaturing. It is assumed that this denaturingis due to the intermediate, unstable monomers (Smith, R. A. andBaglioni, C. (1987), J. Biol. Chem. 262, 6951-6954; Narhi, L. O. andArakawa, T. (1987), Biochem. Biophys. Res. Commun 147, 740-746).

In a first attempt this problem has been addressed in the art forexample in WO 01/25277. WO 01/25277 discloses single-chain TNFαpolypeptides with 3 copies of a TNFα monomer. Due to the single chainnature, dissociation is prevented. A similar concept is disclosed in WO2005/103077.

In addition it has been reported in the prior art that oligomericmolecules of TNF ligand family members exhibit increased activity butlikewise increased toxicity (Koschny R et al. J Mol Med 2007; 85:923-935; Gerspach J et al., Results Probl Cell Differ. 2009; 49:241-73.Review. Wyzgol et al JI 2010).

Thus, there is still in the art a need for novel and improved TNF ligandfamily derived compounds, which are preferably stable, exhibit few(er)or no adverse systemic reactions while in parallel maintainingbiological specificity.

This object is solved by the present invention, in particular by meansas set forth in the appended set of claims and as illustrated in thefollowing.

The inventors of the present invention have surprisingly found thatpolypeptides of the present invention exhibit increased activity againsttumors while they do at the same time surprisingly not exhibit increasedtoxicity towards non-tumor tissue.

Essentially, the polypeptides of the present invention are fusionproteins comprising on the one hand at least three TNF ligand familymember monomers and comprising the variable domains of an antibody V_(L)and V_(H) region linked by a short linker as targeting moiety on theother hand. To increase specificity, to increase in vivo half-life andto modulate pharmacodynamic properties, the polypeptides according tothe present invention may further comprise an albumin binding domainand/or other domains and/or other modifications.

Thus, in a first aspect the present invention relates to a polypeptidewhich comprises:

-   -   a) at least three components A, each of which comprises the        sequence of a TNF homology domain (THD) of a TNF ligand family        member, or functional derivative thereof, and    -   b) at least one component B consisting of a V_(L) region and a        V_(H) region linked directly to each other with a linker        sequence L which has a length of ≤12 amino acid residues.

The term “polypeptide” as used herein refers to a polymer composed of asequence of amino acids. The term shall not be construed as limiting thelength of the polypeptide unit. However, preferably, the polypeptide hasa length of less than 1000 amino acids, more preferably less than 900amino acids. The amino acids within the polymer of said polypeptidesequence are usually linked to each other via peptide bonds, butmodifications of said peptide bond(s) or of side chain residues may betolerable, provided the overall activity is not totally lost, e.g. theresulting chemical entity (e.g. components A) still trimerizes andactivates its targets.

The TNF homology domain is the common structural feature shared by allTNF ligand family members Bodmer J L et al. (Trends Biochem Sci. 2002January; 27(1):19-26.). It comprises the receptor binding sites and isthus critical for the biologic activity of the TNF ligand familymembers. A component A of the present invention may have as minimalmotif the THD, e.g. of a given TNF ligand family member, but may forexample also comprise longer sequence stretches of TNF ligand familymembers such as the sequence of the soluble form (shed or secreted,respectively) of said TNF ligand family member. The sequence may alsocomprise the entire extracellular domain of a TNF ligand family member,but preferably without the protease cleavage site naturally present insome of these TNF ligand family members, e.g. without a TACE/ADAM17cleavage site in order to avoid cleavage of the fusion protein in theregion comprising the three components A.

The THD domain may be for example selected from the TNF ligand familymember group consisting of: FasL (CD95L), TRAIL, TNF, LT alpha, LT beta,CD30L, CD40L, OX40L, RANKL, TWEAK, LIGHT, CD27L, 4-1BBL, GITRL, APRIL,EDA 1, EDA 2, VEGI und BAFF. Particularly preferred are the human TNFligand family members human FasL (CD95L), human TRAIL, human TNF, humanLT alpha, human LT beta, human CD30L, human CD40L, human OX40L, humanRANKL, human TWEAK, human LIGHT, human CD27L, human 4-1 BBL, humanGITRL, human APRIL, human EDA 1, human EDA 2, human VEGI und human BAFF.

Further information, in particular about sequences of the TNF ligandfamily members, may be obtained for example from publicly accessibledatabases such as the GenBank: FasL (CD95L) (GenBank Accession No.NM_000639), TRAIL (TNF Related Apoptosis Inducing Ligand; GenBankAccession No. NM_003810), also Apo2L termed, TNF (Tumor Nekrose Faktor;GenBank Accession No. NM_000594), LT alpha (GenBank Accession No.NM_000595), Lymphotoxin beta (GenBank Accession No. NM_002341), CD30L(CD153; GenBank Accession No. NM_001244), CD40L (CD154; GenBankAccession No. NM_00074), OX40L (GenBank Accession No. NM_003326), RANKL(GenBank Accession No. NM_003701), TWEAK (GenBank Accession No.NM_003809), LIGHT (GenBank Accession No. NM_003807), CD27L (GenBankAccession No. NM_001252), 4-1 BBL (GenBank Accession No. NM_003811),GITRL (GenBank Accession No. NM_005092), APRIL (GenBank Accession No.NM_172089), EDA 1/2 (GenBank Accession No. NM_001399; NM_001005609),VEGI (GenBank Accession No. NM_005118) und BAFF (GenBank Accession No.NM_006573).

The sequences of the at least three components A of the polypeptidesaccording to the invention may be selected independently of each other;e.g. the three components A may have the respective sequence of the sameTNF ligand family member or may have the sequence of different TNFligand family members or 2 of them may be identical while the other onediffers in sequence (in terms of length and/or sequence). THD domains ofdifferent TNF ligand family members is in particular possible if the THDdomain is selected from LT alpha or LT beta. Otherwise, it isparticularly preferred if all three components A comprise the THD of thesame TNF ligand family member. Certainly, similar considerations applyif the polypeptide according to the present invention comprises morethan 3 components A. For example, the inventive polypeptide may comprise4, 5, 6 or more components A. If the polypeptide according to thepresent invention comprises more than 3 components A then it isparticularly preferred that the polypeptide comprises a multiple ofthree components A. By this means, two, three, four or moreconsecutively arranged trimers may be formed.

In a preferred embodiment a given component A may comprise or consist ofone of the human sequences according to SEQ ID NOs.: 1-38 as indicatedin Table 1 below, or functional fragments or functional derivativesthereof, which includes natural or artificial variations thereof orrespective orthologs from other species. Preferred are orthologs fromother mammalian species such as chimpanzee, mouse, swine, rat etc.

TABLE 1 Possible Components A AA SEQ ID Name: Position NO: SequenceTRAIL 120-281  1 QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFY YIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQ GGIFELKENDRIFVSVTNEHLIDMDHEASFFG AFLVGTRAIL 118-281  2 GPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEK GFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSI YQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG TRAIL 116-281  3 ERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHE KGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYS IYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG TRAIL 114-281  4 VRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVI HEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYG LYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG TRAIL  95-281  5 TSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESS RSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPIL LMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG mouse TRAIL  99-291  6 TFQDTISTVPEKQLSTPPLPRGGRPQKVAAHI TGITRRSNSALIPISKDGKTLGQKIESWESSRKGHSFLNHVLFRNGELVIEQEGLYYIYSQTYFR FQEAEDASKMVSKDKVRTKQLVQYIYKYTSYPDPIVLMKSARNSCWSRDAEYGLYSIYQGG LFELKKNDRIFVSVTNEHLMDLDQEASFFGA FLINFasL (CD95L) 144-281  7 RKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNL PLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVS ELSLVNFEESQTFFGLYKL FasL (CD95L) 142-281 8 ELRKVAHLTGKSNSRSMPLEWEDTYGIVLLS GVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMM SYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL FasL (CD95L) 137-281  9PPEKKELRKVAHLTGKSNSRSMPLEWEDTYG IVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGK MMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL FasL (CD95L) 130-281 10QIGHPSPPPEKKELRKVAHLTGKSNSRSMPLE WEDTYGIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDL VMMEGKMMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL FasL (CD95L) 120-281 11QMHTASSLEKQIGHPSPPPEKKELRKVAHLT GKSNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKV YMRNSKYPQDLVMMEGKMMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNF EESQTFFGLYKL Mouse FasL 137-279 12EKKEPRSVAHLTGNPHSRSIPLEWEDTYGTA (CD95L) LISGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNQPLNHKVYMRNSKYPEDLVLMEEKRL NYCTTGQIWAHSSYLGAVFNLTSADHLYVNISQLSLINFEESKTFFGLYKL TNF  89-233 13 VAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPS THVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEI NRPDYLDFAESGQVYFGIIAL TNF  77-233 14VRSSSRTPSDKPVAHVVANPQAEGQLQWL NRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKV NLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGII AL LT alpha  59-205 15SNLKPAAHLIGDPSKQNSLLWRANTDRAFL QDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSS QKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL LT beta  82-244 16DLSPGLPAAHLIGAPLKGQGLGWETTKEQA FLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYG PGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFA RGKTFFGAVMVG LT beta 86-244 17GLPAAHLIGAPLKGQGLGWETTKEQAFLTS GTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTP ELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARG KTFFGAVMVG CD30L  97-234 18KSWAYLQVAKHLNKTKLSWNKDGILHGVR YQDGNLVIQFPGLYFIICQLQFLVQCPNNSVDLKLELLINKHIKKQALVTVCESGMQTKHV YQNLSQFLLDYLQVNTTISVNVDTFQYIDTSTFPLENVLSIFLYSNSD CD30L 102-234 19 LQVAKHLNKTKLSWNKDGILHGVRYQDGNLVIQFPGLYFIICQLQFLVQCPNNSVDLKLE LLINKHIKKQALVTVCESGMQTKHVYQNLSQFLLDYLQVNTTISVNVDTFQYIDTSTFPLEN VLSIFLYSNSD CD40L 116-261 20GDQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRA ANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL CD40L 113-261 21 MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGA SVFVNVTDPSQVSHGTGFTSFGLLKL OX40L  52-18322 VSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDE IMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKD KVYLNVTTDNTSLDDFHVNGGELILIHQNP GEFCVLOX40L  55-183 23 RYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHY QKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEF CVL RANKL 161-317 24EAQPFAHLTINATDIPSGSHKVSLSSWYHDR GWAKISNMTFSNGKLIVNQDGFYYLYANICFRHHETSGDLATEYLQLMVYVTKTSIKIPSSH TLMKGGSTKYWSGNSEFHFYSINVGGFFKLRSGEEISIEVSNPSLLDPDQDATYFGAFKVRD ID RANKL 140-317 25IRAEKAMVDGSWLDLAKRSKLEAQPFAHLTI NATDIPSGSHKVSLSSWYHDRGWAKISNMTFSNGKLIVNQDGFYYLYANICFRHHETSGDL ATEYLQLMVYVTKTSIKIPSSHTLMKGGSTKYWSGNSEFHFYSINVGGFFKLRSGEEISIEVSNP SLLDPDQDATYFGAFKVRDID TWEAK 94-249 26SAPKGRKTRARRAIAAHYEVHPRPGQDGA QAGVDGTVSGWEEARINSSSPLRYNRQIGEFIVTRAGLYYLYCQVHFDEGKAVYLKLDLLV DGVLALRCLEEFSATAASSLGPQLRLCQVSGLLALRPGSSLRIRTLPWAHLKAAPFLTYFGLF QVH TWEAK 105-249 27RAIAAHYEVHPRPGQDGAQAGVDGTVSG WEEARINSSSPLRYNRQIGEFIVTRAGLYYLYCQVHFDEGKAVYLKLDLLVDGVLALRCLEEF SATAASSLGPQLRLCQVSGLLALRPGSSLRIRTLPWAHLKAAPFLTYFGLFQVH LIGHT  83-240 28 LIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYI YSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGG VVHLEAGEKVVVRVLDERLVRLRDGTRSYF GAFMVCD27L  51-193 29 ESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHI QVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLT GTLLPSRNTDETFFGVQWVRP CD27L  56-193 30DVAELQLNHTGPQQDPRLYWQGGPALGR SFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSF HQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP 4-1 BBL  85-254 31 LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVF FQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHL SAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE GITRL  50-177 32 QLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYN DVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILL ANPQFIS APRIL 112-250 33KKQHSVLHLVPINATSKDDSDVTEVMWQP ALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRS MPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKL EDA-1 245-391 34 ENQPAVVHLQGQGSAIQVKNDLSGGVLNDWSRITMNPKVFKLHPRSGELEVLVDGTYFI YSQVEVYYINFTDFASYEVVVDEKPFLQCTRSIETGKTNYNTCYTAGVCLLKARQKIAVKMV HADISINMSKHTTFFGAIRLGEAPAS EDA-2 245-38935 ENQPAVVHLQGQGSAIQVKNDLSGGVLN DWSRITMNPKVFKLHPRSGELEVLVDGTYFIYSQVYYINFTDFASYEVVVDEKPFLQCTRSIE TGKTNYNTCYTAGVCLLKARQKIAVKMVHADISINMSKHTTFFGAIRLGEAPAS VEGI 72-251 36 LKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLAFTK NRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPT QLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL VEGI 93-251 37DKPRAHLTVVRQTPTQHFKNQFPALHWEH ELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVT DSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFF GAFLL BAFF 134-285 38AVQGPEETVTQDCLQLIADSETPTIQKGSYT FVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLV TLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL

The column “Amino acid position” indicates the amino acid residues inthe full length native protein, i.e. the sequence given in the sequencecolumn corresponds to the respective amino acid residues in the fulllength sequence. For example, SEQ ID NO: 1 corresponds to amino acidresidues 120-281 of full length human TRAIL.

Information about natural variations of SEQ ID NOs: 1-38 and respectiveorthologs from other species may be easily obtained from publiclyaccessible databases comprising information about proteins of the TNFligand family members or respective nucleic acid sequences. Examples forsuch databases are UniProt; SwissProt, TrEMBL, Protein InformationResource (PIR); Genbank, EMBL-Bank; DNA data bank of Japan (DDBJ) etc.Orthologs of other species may in particular be likewise identified viae.g. BLAST searches on basis of the respective SEQ ID NOs: 1-38.

Preferred substitutions in human TRAIL in this regard affect at leastone of the following amino acids of human TRAIL: R130, G160, Y189, R191,Q193, E195, N199, K201, Y213, T214, S215, H264, 1266, D267, D269.Preferred amino acid substitutions of human TRAIL are at least one ofthe following substitutions: R130E, G160M, Y189A, Y189Q, R191K, Q193S,Q193R, E195R, N199V, N199R, K201R, Y213W, T214R, S215D, H264R, 1266L,D267Q, D269H, D269R, or D269K. Double or multiple substitutions are alsopossible, e.g. Y213W/S215D; E195R/D269H, T214R/D269H;Q193S/N199V/K201R/Y213W/S215D. Functional mutants of TRAIL are forexample described in R. F. Kelley et al. (J. Biol. Chem.; 280 (2005)2205-2212) M. MacFarlane et al. (Cancer Res 65 (2005) 11265-11270), A.M. van der Sloot et al. (Proc. Natl. Acad. Sci. USA 103 (2006)8634-8639), V. Tur et al. (J. Biol. Chem. 283 (2008) 20560-20568), andGasparian et al. (Apoptosis 14 (2009) 778-787). Functional mutants ofTRAIL are in particular TRAIL (96-281), TRAIL(96-281)-Y189N/R191K/Q193R/H264R/1266L/D267Q, TRAIL(96-281)-Y189A/Q193S/N199V/K201/Y213W/S215D, TRAIL(96-281)-Q193S/N199V/K201R/Y213W/S215N, TRAIL(96-281)-Y189Q/R191K/Q193R/H264R/I266L/D267Q, TRAIL (114-281), TRAIL(114-281)-D269H/E195R, TRAIL (114-281)-D269H, TRAIL (114-281)-D218H,TRAIL (114-281)-D218Y, TRAIL(114-281)-Y189A/Q193S/N199V/K201/Y213W/S215D, TRAIL(114-281)-Y189N/R191K/Q193R/H264R/I266LD267Q, TRAIL(114-281)-Y189N/R191K/Q193R/H264R/I266L/D267Q/D269H, and TRAIL(114-281)-Y189N/R191K/Q193R/H264R/I266L/D269H.

As mentioned above, a component A as used herein refers to a polypeptidecomprising the sequence of a TNF homology domain (THD); or a functionalderivative thereof. Since the polypeptides of the present inventioncomprise three of said components A, trimer formation and thus theformation of the active conformation is possible. A polypeptideaccording to the present invention furthermore exhibits due to thepresence of the at least 3 components A binding activity for the bindingpartners of TNF ligand family members, such as membrane-bound receptors.Functional derivatives of the TNF ligand family member sequence mayexhibit (slightly) different activities and biological functions, e.g.regarding specificity or selectivity, but their overall biologicalfunction is maintained. In particular, the receptor binding activityshould be maintained.

In the prior art, a multitude of methods is disclosed which allow theassessment of the biological activity of a protein, polypeptide or othermolecule, respectively. Examples are protein analytical methods such asImmunoblot, ELISA, Radioimmunoassay, Immune precipitation, SurfacePlasmon Resonance (Biacore), Quartz Crystal Microbalance (QCM); cell andtissue analytical methods such as immunocytochemistry,immunohistochemistry, fluorescence microscopy, FACS; cell functionassays: such as cytokine-release assays, proliferation and cell cycleassays (³H-thymidine incorporation, CFSE staining), cytotoxicity assays,apoptosis assays, NFkB bandshift (EMSA) and reporter gene (Luciferase)assays, kinase assays (e.g. in Antibodies: a laboratory manual. Harlow &Lane, Cold Spring Harbor Laboratory Press; 1 edition (Dec. 1, 1988)Current Protocols in Immunology, Wiley and Sons, 1992); Cell Biology, ALaboratory Handbook 3rd ed., J Celis et al, eds. Elsevier, 2006). Bythis means a person skilled in the art will readily be able to assesswhether a functional fragment or functional derivative of the solubleTNF ligand family member sequence retains the overall properties of thesoluble TNF ligand family member (e.g. induction of apoptosis/cell deathor activation of NFkB).

The term “derivative”, e.g. a functional derivative of the THD sequenceor of one of the sequences according to SEQ ID NOs.: 1-38, is intendedto refer also to sequences which exhibit a functional and structuralsimilarity to the respective reference sequence. In particular therespective derivative will preferably exhibit a sequence identity of atleast 50%, more preferably at least 60%, more preferably at least 70%,more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 92%, more preferablyat least 94%, more preferably at least 95%, more preferably at least96%, more preferably at least 97%, more preferably at least 98%, andmost preferably at least 99% sequence identity with the respectivereference sequence. A person skilled in the art will understand thatsuch levels of sequence identity are preferably less than 100%. Thederivative sequence and the reference sequence may differ from eachother in terms of one or more insertions, deletions and/orsubstitutions.

As used herein, the term “% sequence identity”, has to be understood asfollows: Two sequences to be compared are aligned to give a maximumcorrelation between the sequences. This may include inserting “gaps” ineither one or both sequences, to enhance the degree of alignment. A %identity may then be determined over the whole length of each of thesequences being compared (so-called global alignment), that isparticularly suitable for sequences of the same or similar length, orover shorter, defined lengths (so-called local alignment), that is moresuitable for sequences of unequal length. In the above context, an aminoacid sequence having a “sequence identity” of at least, for example, 95%to a query amino acid sequence, is intended to mean that the sequence ofthe subject amino acid sequence is identical to the query sequenceexcept that the subject amino acid sequence may include up to five aminoacid alterations per each 100 amino acids of the query amino acidsequence. In other words, to obtain an amino acid sequence having asequence of at least 95% identity to a query amino acid sequence, up to5% (5 of 100) of the amino acid residues in the subject sequence may beinserted or substituted with another amino acid or deleted.

Methods for comparing the identity of two or more sequences are wellknown in the art. The percentage to which two sequences are identicalcan for example be determined by using a mathematical algorithm. Apreferred, but not limiting, example of a mathematical algorithm whichcan be used is the algorithm of Karlin et al. (1993), PNAS USA,90:5873-5877. Such an algorithm is integrated in the BLAST family ofprograms, e.g. BLAST or NBLAST program (see also Altschul et al., 1990,J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res,25:3389-3402), accessible through the home page of the NCBI at worldwide web site ncbi.nlm.nih.gov) and FASTA (Pearson (1990), MethodsEnzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci.U.S.A 85, 2444-2448). Sequences which are identical to other sequencesto a certain extent can be identified by these programmes. Furthermore,programs available in the Wisconsin Sequence Analysis Package, version9.1 (Devereux et al., 1984, Nucleic Acids Res., 387-395), for examplethe programs BESTFIT and GAP, may be used to determine the % identitybetween two polypeptide sequences. BESTFIT uses the “local homology”algorithm of (Smith and Waterman (1981), J. Mol. Biol. 147, 195-197) andfinds the best single region of similarity between two sequences.

Functional derivatives in component A may in particular exhibitselective receptor binding properties, or may be optimized regardingbioactivity or other properties such as stability. In particular suchderivatives may exhibit altered sequences at protease cleavage sites.

Derivatives as used herein in particular include those amino acidsequences which exhibit (for example in the context of a given level ofsequence identity) in view of the respective reference sequenceconservative substitutions. Conservative amino acid substitutions arepreferably considered to occur within a group of amino acid residueswhich have sufficiently similar physicochemical properties, so that asubstitution between members of the group will preserve the biologicalactivity of the molecule (see e.g. Grantham, R. (1974), Science 185,862-864). Particularly, conservative amino acid substitutions arepreferably substitutions in which the amino acids originate from thesame class of amino acids (e.g. basic amino acids, acidic amino acids,polar amino acids, amino acids with aliphatic side chains, amino acidswith positively or negatively charged side chains, amino acids witharomatic groups in the side chains, amino acids the side chains of whichcan enter into hydrogen bridges, e.g. side chains which have a hydroxylfunction, etc.). Conservative substitutions are in the present case forexample substituting a basic amino acid residue (Lys, Arg, His) foranother basic amino acid residue (Lys, Arg, His), substituting analiphatic amino acid residue (Gly, Ala, Val, Leu, lie) for anotheraliphatic amino acid residue, substituting an aromatic amino acidresidue (Phe, Tyr, Trp) for another aromatic amino acid residue,substituting threonine by serine or leucine by isoleucine. Furtherconservative amino acid exchanges will be known to the person skilled inthe art.

Insertions, deletions and substitutions are in particular at suchsequence positions possible where they do not induce a change in thethree-dimensional structure or where they do not affect the bindingregion. A change in three-dimensional structure by means of insertionsor deletions may for example be verified with CD spectral analysis(circular dichroism) (Urry 1985, Absorption, Circular Dichroism and ORDof Polypeptides, in: Modern Physical Methods in Biochemistry, Neubergeret al. (Eds.), Elsevier, Amsterdam, NL).

Suitable methods for producing derivatives of polypeptide sequencesaccording to the present invention or components thereof, which exhibitin view of the reference sequence a substitution, are for exampledisclosed in U.S. Pat. Nos. 4,737,462, 4,588,585, 4,959,314, 5,116,943,4,879,111 and 5,017,691. The production of derivatives as used herein isdescribed in general in Maniatis et al. (2001), Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press. For thisapproach individual codons may simply be skipped, added or exchanged.Derivatives of polypeptide sequences mentioned herein may in particularbe such polypeptide sequences which are stabilized vis-à-vis therespective reference sequence and which are less prone to physiologicaldegradation. An example for such modification is the stabilization ofthe protein backbone by substitution of the amid-like bonds by usinge.g. g-amino acids.

Derivatives of sequences according to the invention may in particular beproduced by introducing changes into the nucleic acid sequence whichencode the respective polypeptide reference sequence. Such changes maybe insertions, deletions and/or substitutions of one or morenucleotides, preferably without induction of a frame shift. In the art,a multitude of methods is known for introducing such changes to nucleicacid sequences. A most common technique is the oligonucleotide-directedsite-specific mutagenesis (see Comack B., Current Protocols in MolecularBiology, 8.01-8.5.9, Ausubel F. et al., 1991). Briefly, anoligonucleotide is synthesized which exhibits the sequence of a specificmutation. This oligonucleotide is then hybridized with the template(reference sequence). Preferably, this technique is used for asingle-stranded template. After annealing of the modifiedoligonucleotide and the template, a DNA-dependent DNA polymerase isadded in order to synthesize the second strand of the oligonucleotidewhich is complementary to the templated DNA strand. As a result, aheteroduplex molecule is formed, which comprises a mismatch, which isdue to the above-mentioned mutation in the oligonucleotide. Theoligonucleotide sequence is then introduced in a suitable plasmid whichis in turn introduced into a suitable host cell. In the host cell theoligonucleotide is then replicated. By this means a nucleic acidsequence is obtained with specific changes (mutations) which may be usedfor the production of derivatives according to the present invention.

In a preferred embodiment of the present invention all and/or at leastthree components A of the polypeptide according to the present inventionhave the sequence of SEQ ID NO: 1 and/or SEQ ID NO: 5.

Preferably, the at least three components A of the polypeptidesaccording to the present invention are linked to each other by at leasttwo intervening peptide linkers P. In other words, two given componentsA within the polypeptide according to the present invention arepreferably linked to each other directly via a peptide linker (e.g.A-P-A-P-A). Peptide linkers P are preferably flexible amino acidstretches and/or do not affect the intrinsic trimerization properties ofthe components A within the polypeptide according to the presentinvention. Preferably, such peptide linkers P are less than 50, evenmore preferably less than 45, even more preferably less than 40, evenmore preferably less than 35, even more preferably less than 30, evenmore preferably less than 25, even more preferably less than 20, evenmore preferably less than 15, even more preferably less than 10 aminoacids long. Alternatively or in addition, the peptide linkers P havepreferably an amino acid length of 1 amino acid or more, 2 amino acidsor more, 3 amino acids or more, 4 amino acids or more, 5 amino acids ormore, 6 amino acids or more, 7 amino acids or more, and/or 8 amino acidsor more. A peptide linker linking two components A of a polypeptide ofthe present invention may thus have for example an amino acid length inthe range of 2 to 50 amino acids, 2 to 30 amino acids, 3 to 25 aminoacids, 4 to 16 amino acids, 4 to 12 amino acids or any other combinationof amino acids lengths disclosed above for peptide linkers. Particularlypreferred are peptide linker lengths of 1 to 8 amino acids, e.g. 4 or 8amino acids.

In terms of amino acid sequence, the peptide linkers P linking thecomponents A within the polypeptide of the present invention arepreferably glycine (G) rich peptide linkers, i.e. are amino acidsequences with a high glycine content of more than 50%; e.g. from atleast 60 to 80%, for example of about 75%. Other amino acids which maybe present in the peptide linker are for example serine residues or lesspreferably alanine residues or glutamine residues. The peptide linker Pmay be composed of repetitive units. For example the linker may compriseseveral units of GG (SEQ ID NO: 40); GGS (SEQ ID NO: 55); GSG (SEQ IDNO: 54), or SGG (SEQ ID NO: 53) and combinations thereof. The peptidelinker may also be of type which may easily be modified, e.g.glycosylated. An example for such sequence are SEQ ID NOs: 83-91.Particularly preferred examples for a peptide linker P linking twocomponents A of the present invention are selected from the group ofsequences as depicted in Table 2 below:

TABLE 2 Possible Peptide linkers SEQ ID Name: Length NO: Sequence (G)₁ 1 39 G (G)2  2 40 GG (G)3  3 41 GGG (G)4  4 42 GGGG (G)5  5 43 GGGGG(G)6  6 44 GGGGGG (G)₇  7 45 GGGGGGG (G)₈  8 46 GGGGGGGG GGGS  4 47 GGGS(GGGS)₂  8 48 GGGSGGGS (GGGS)₃ 12 49 GGGSGGGSGGGS GGGGS  5 50 GGGGS(GGGGS)₂ 10 51 GGGGSGGGGS (GGGGS)₃ 15 52 GGGGSGGGGSGGGGS (SGG)₁  3 53SGG (GSG)₁  3 54 GSG (GGS)₁  3 55 GGS (SGGG)₁  4 56 SGGG (GSGG)₁  4 57GSGG (GGSG)₁  4 58 GGSG (SGGGG)₁  5 59 SGGGG (GSGGG)₁  5 60 GSGGG(GGSGG)₁  5 61 GGSGG (GGGSG)₁  5 62 GGGSG (SGG)₂  6 63 SGGSGG (GSG)₂  664 GSGGSG (GGS)₂  6 65 GGSGGS (SGGG)₂  8 66 SGGGSGGG (GSGG)₂  8 67GSGGGSGG (GGSG)₂  8 68 GGSGGGSG (SGGGG)₂ 10 69 SGGGGSGGGG (GSGGG)₂ 10 70GSGGGGSGGG (GGSGG)₂ 10 71 GGSGGGGSGG (GGGSG)₂ 10 72 GGGSGGGGSG (SGG)₃  973 SGGSGGSGG (GSG)₃  9 74 GSGGSGGSG (GGS)₃  9 75 GGSGGSGGS (SGGG)₃ 12 76SGGGSGGGSGGG (GSGG)₃ 12 77 GSGGGSGGGSGG (GGSG)₃ 12 78 GGSGGGSGGGSG(SGGGG)₃ 15 79 SGGGGSGGGGSGGGG (GSGGG)₃ 15 80 GSGGGGSGGGGSGGG (GGSGG)₃15 81 GGSGGGGSGGGGSGG (GGGSG)₃ 15 82 GGGSGGGGSGGGGSG N-Glyco  9 83GNGTSNGTS N-Glyco (1)  9 84 GNGTSNGTG N-Glyco (2)  9 85 GNGTSNGTSGN-Glyco (3)  9 86 GNGTSNGTGS N-Glyco (4) 13 87 GNGTSNGTSNGTS N-Glyco (5)13 88 GGGSGNGTSNGTGS N-Glyco (6) 13 89 GNGTSNGTGSGGGS N-Glyco (7) 13 90GGGSGNGTSNGTSG N-Glyco (8) 13 91 GNGTSNGTSGGGGS

A person skilled in the art will understand that the above mentionedlinker peptides may also be combined and a multitude of other flexiblelinker sequences may be utilized in similar manner as long as they dopreferably not interfere with the trimeric assembly of component A. SEQID NO: 48, 88 and 90 are particularly preferred as peptide linker Plinking two components A of the polypeptide according to the presentinvention. Preferably the peptide linkers P linking the components Awithin the polypeptide of the present invention do not comprise anycysteine residues in order to avoid formation of intramoleculardisulfide bridges which could negatively impact the trimer formation ofthe components A.

The at least two peptide linkers P linking the least three components Aof the polypeptides according to the present invention may be inprinciple selected independently of each other; e.g. the at least twopeptide linkers P may have the same sequence or may have differentsequences (in terms of length and/or sequence). However, it isparticularly preferred if the peptide linkers P linking the at leastthree components A are identical. Certainly, similar considerationsapply if the polypeptide according to the present invention comprisesmore than 3 components A and more than two peptide linkers P linkingsaid components A. The peptide linkers P are linked to the components Avia a covalent bond to the C-terminus of a first component A and acovalent bond to the N-terminus of the subsequent component A.Preferably, the linkages are peptide bonds.

Specific examples of polypeptides according to the present inventioncomprise as components A SEQ ID NO: 5 and preferably SEQ ID NOs: 48, 88and/or 90 as peptide linkers P.

As mentioned above, the polypeptides according to the present inventioncomprise alongside the at least three components A at least onecomponent B consisting of a V_(L) region and a V_(H) region linkeddirectly to each other with a linker sequence L which has a length of≤12 amino acids.

The terms “V_(L)” and “V_(H)” refer to the V_(L) and V_(H) regions of anantibody, i.e. the N-terminal variable region of the light chain of animmunoglobulin and the N-terminal variable region of the heavy chain ofan immunoglobulin, respectively. Both terms are well understood in theart and are structurally well defined. The individual V_(L) and V_(H)regions are each composed of 3 hypervariable regions (CDR1, CDR2, CDR3;CDR: complementarity determining region) and 4 framework regions (FR1,FR2, FR3, FR4). Identifying the respective subregions within a givensequence is routine in the art and may for example be accomplished byIgBlast of the NCBI, v-base of the MRC, v-base2 hosted by EU-GENE,on-line programs provided by the group of Andrew Martin and/or theExPASy Proteomics Server. Kabat nomenclature may also be useful (Martin,A. C. R. PROTEINS: Structure, Function and Genetics, 25 (1996),130-133). The variable regions of the heavy and the light chain formtogether the binding region of an antibody. In immunoglobulins, theV_(L) and V_(H) regions are on different polypeptide chains. In thepolypeptides of the present invention the V_(L) and V_(H) regions are onthe same chain. Interaction of a V_(L) domain with a V_(H) domain(intra- or intermolecularly) allows the polypeptide of the presentinvention to bind to the respective target antigen.

Preferably, the V_(L) and the V_(H) region of the polypeptide accordingto the present invention are V_(L) and V_(H) regions of an antibodybinding (preferably) specifically to a cell surface molecule (cellsurface antigen), in particular to a cell surface molecule selected fromthe group consisting of: a cytokine receptor, a growth factor receptor,an integrin, a cell adhesion molecule and/or a cell type- ortissue-specific cell surface antigen, cell surface expressedtumor-associated antigens (TAA), carbohydrates. A tumor-associatedantigen may for example be expressed on tumor cells per se, on malignantcells, on stroma cells, on tumor endothelium and other tumor localizedcell types.

In a preferred embodiment the V_(L) and the V_(H) region of thepolypeptide according to the present invention are V_(L) and V_(H)regions of an antibody binding to a target antigen selected from thegroup consisting of: the erbB family of tyrosine kinase receptors (EGFR,HER2, HER3, HER4), VEGFRs, hetermeric integrin a_(x) β_(x) receptorfamily, fibroblast activation protein (FAP), galectin, EpCAM, CEA, CD44and tumor specific variants thereof (CD44v) and other tumor selectivecell surface markers, CD2, CD5, CD7, CD19, CD20, CD21, CD22, CD24, CD25,CD30, CD33, CD38, CD40, CD52, CD56, CD71, CD72, CD73, CD105, CD117,CD123, c-Met, PDGFR, IGF1-R, HMW-MAA, TAG-72, GD2, GD3, GM2, folatereceptor, Le^(y), MUC-1, MUC-2, PSMA, PSCA, uPAR, Claudin 18.2, etc.Particularly preferred targets are the members of the erbB family oftyrosine kinase receptors and tumor stroma selective targets such asFAP.

As proof of concept, EGFR is used as target antigen in the appendedexamples.

Respective V_(H) and V_(L) sequences may easily be obtained by a personskilled in the art. Polypeptide and nucleic acid sequences for manyantibodies are readily available in the art (see for example Expasysequence database, PubMed etc.). Alternatively, a person skilled in theart may determine the sequence of available antibodies of the desiredspecificity or may even produce a new antibody against the desiredtarget antigen by immunizing an animal suitable for antibody productionwith the antigen, isolating antigen specific B-cell clones andsequencing the respective V_(L) and V_(H) genes. Antibodies (andsubsequently antibody sequences) may also be obtained from recombinantantibody libraries, e.g. immune, naive, semi-synthetic or fullysynthetic antibody libraries. Isolation from such libraries can beachieved by different means, e.g. by phage display, ribosome display,yeast display, bacterial display, high-throughput screening, etc. Bymeans of genetic engineering said sequences may then be included in anucleic acid sequence encoding a polypeptide of the present invention.

A person skilled in the art will understand that the V_(H) and V_(L)regions in the polypeptide according to the present invention may beartificial, i.e. need not be derived from a de facto naturally occurringantibody. Rather this terminology is intended to reflect that saidregions exhibit the general architecture of V_(L) and V_(H) regions. TheV_(L) and V_(H) regions of the polypeptides according to the presentinvention may for example be humanized sequences, e.g. while the CDRsare of mouse origin, the framework regions are of human origin. TheV_(L) and V_(H) regions may be for example deimmunized and/or fullyhuman.

A particularly preferred V_(L) region is SEQ ID NO: 92 if the targetantigen is EGFR (see for example in SEQ ID NOs: 102 and 107).

A particularly preferred V_(H) region is SEQ ID NO: 93 if the targetantigen is EGFR (see for example in SEQ ID NOs: 102 and 107).

Particularly preferred V_(L) regions if the target antigen is FAP arethe amino acid sequences according to SEQ ID NOs: 130-132 (see forexample in SEQ ID NOs: 127-129).

Particularly preferred V_(H) regions if the target antigen is FAP arethe amino acid sequences according to SEQ ID NOs: 133-135 (see forexample in SEQ ID NOs: 127-129).

The TRAIL fusion proteins according to SEQ ID NOs: 127-129 are examplesof tumorstroma targeted TRAIL fusion proteins recognizing the selectivetumorstroma marker fibroblast activation protein (FAP). Thus, TRAILproapoptotic activity is directed to the tumor environment and throughjuxtatropic presentation of the TRAIL module within the fusion protein,apoptosis is signaled in trans to the tumor cell. Highly specificapoptotic activity of the TRAIL module ensures efficient antitumoraltherapeutic action towards a wide variety of carcinomas, as FAPoverexpression is a prominent and common feature of a variety ofepithelial cancers, including breast, colon, pancreas and lung, with avariable stroma content comprising 10 to 90% of total tumor mass (GarinChesa et al, 1990, PNAS 87:7235-7239).

The fusion proteins according to SEQ ID NOs: 127-129 are FAP specificand exhibit high binding affinity typically in the nanomolar range (e.g.10-30 nM). The components B of the fusion proteins according to SEQ IDNOs: 127-129 are 1) a humanized variant, generated by CDR grafting, andbeing species crossreactive between mouse and human (SEQ ID NOs: 130,133, 136 and 127), thus allowing preclinical studies in murine tumormodels; 2) two fully human components B, isolated by guided selectionfrom a naïve human Ig library (SEQ ID NOs: 131, 134, 137 and 128, andSEQ ID NOs: 132, 135, 138 and 129) and binding to different epitopes atthe extracellular domain of human FAP, one characterized by competitionwith the murine mab F19 (SEQ ID NOs: 131, 134, 137 and 128), the otherone not competing with F19 for FAP binding (SEQ ID NOs: 132, 135, 138and 129).

The V_(L) region and the V_(H) region may be arranged in any suitablemanner in the polypeptide according to the present invention.Preferably, the region comprising the V_(L) region and the V_(H) regionis arranged N-terminally of the region comprising the three componentsA.

As mentioned above, the V_(L) region and a V_(H) region of component Bof the polypeptides according to the present invention are linkeddirectly to each other with a linker sequence L which has a length of≤12 amino acids. Linker sequence L is preferably a flexible amino acidstretch. Preferably, such linker sequence L is less than 11, even morepreferably less than 10, even more preferably less than 9, even morepreferably less than 8, even more preferably less than 7, even morepreferably less than 6 amino acids long. In addition, the linkersequence L may have preferably an amino acid length of 0 amino acids ormore, 1 amino acid or more, 2 amino acids or more, 3 amino acids ormore, 4 amino acids or more, 5 amino acids or more, 6 amino acids ormore, 7 amino acids or more, and/or 8 amino acids or more. A linkersequence L linking the V_(L) and V_(H) region of a component B of apolypeptide of the present invention may thus have for example an aminoacid length in the range of 0 to 12 amino acids, 1 to 12 amino acids, 2to 10 amino acids, 3 to 10 amino acids, 3 to 9 amino acids, 3 to 6 aminoacids, 4 to 8 amino acids, 4 to 7 amino acids, 4 to 12 amino acids orany other combination of amino acids lengths disclosed above for linkersequence L. Particularly preferred are linker sequence L lengths of 0 to5 amino acids, in particular 5 amino acids.

In terms of amino acid sequence, the linker sequence L linking the V_(L)and V_(H) region of a component B of a polypeptide of the presentinvention is preferably a glycine (G) rich peptide linker, i.e. has anamino acid sequences with a high glycine content of more than 50%; e.g.from at least 60 to 90%, for example of about 80%. Other amino acidswhich may be present in the linker sequence L are for example serineresidues or less preferably alanine residues or glutamine residues. Thelinker sequence L may be composed of repetitive units. For example thelinker may comprise two, three or four units of GG (SEQ ID NO: 40); GGS(SEQ ID NO: 55); GSG (SEQ ID NO: 54), or SGG (SEQ ID NO: 53) andcombinations thereof. Particularly preferred examples for a linkersequence L linking the V_(L) and V_(H) region of a component B of apolypeptide of the present invention are selected from the group ofsequences as depicted in Table 2 above (except for those sequencesexceeding the length restriction of linker sequence L. Thus, if a linkersequence L is present, the linker sequence L may for example be selectedfrom linker sequences of SEQ ID NOs. 39-51, and 53-78.

SEQ ID NO: 50 is particularly preferred as linker sequence L linking theV_(L) and V_(H) region of a component B of a polypeptide of the presentinvention. Preferably the linker sequence L does not comprise anycysteine residues in order to avoid formation of intramoleculardisulfide bridges, which could for example negatively impact the correctformation of the V_(H) or V₁ secondary structure of the polypeptideaccording to the present invention.

Linker sequence L may certainly be selected independently for eachcomponent B present in the polypeptide according to the presentinvention and independently of any peptide linker P selected for linkingthe components A. Linker sequence L links the V_(L) region and the V_(H)region via a covalent bond. Preferably the linkages are peptide bonds.Component B may have (from N- to C-Terminus) the sequence:

-   -   V_(L) region-linker sequence L-V_(H) region        or may have the sequence    -   V_(H) region-linker sequence L-V_(L) region.

The arrangement V_(H) region-linker sequence L-V_(L) region isparticularly preferred.

In a particularly preferred embodiment, component B has the sequence ofSEQ ID NO: 94 which is composed of SEQ ID NOs: 93, 50 and 92 (see forexample in SEQ ID NOs: 102 and 107).

In this context the present invention also relates to a polypeptidecomprising the sequence of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 92and SEQ ID NO: 93, and/or SEQ ID NO: 94.

In further particularly preferred embodiments, component B has thesequence of any of SEQ ID NOs: 136-138 which are composed of SEQ ID NOs:133, 50 and 130 (see for example in SEQ ID NO: 127), SEQ ID NOs: 134, 50and 131 (see for example in SEQ ID NO: 128), and SEQ ID NOs: 135, 50 and132 (see for example in SEQ ID NO: 129), respectively.

In this context the present invention also relates to a polypeptidecomprising the sequence according to SEQ ID NO: 130, SEQ ID NO: 133, SEQID NO: 130 and SEQ ID NO: 133, and/or SEQ ID NO: 136, to a polypeptidecomprising the sequence according to SEQ ID NO: 131, SEQ ID NO: 134, SEQID NO: 131 and SEQ ID NO: 134, and/or SEQ ID NO: 137, and to apolypeptide comprising the sequence according to SEQ ID NO: 132, SEQ IDNO: 135, SEQ ID NO: 132 and SEQ ID NO: 135, and/or SEQ ID NO: 138.

It will be understood by a person skilled in the art that a component Bof a polypeptide according to the present invention does not compriseany antibody constant regions such as in Fab fragments.

Polypeptides according to the present invention comprise at least threecomponents A and at least one component B. Preferably, the regioncomprising the at least three components A is linked to component B viaa peptide linker X. Peptide linker X can in principle be any sequence aslong as it does neither interfere with formation of the THD trimer norwith the association of a V_(H) domain with a V_(L) domain. Inparticular, there is no absolute length restriction with regard tolinker X and no requirement for flexibility. However, preferably X isless than 50, even more preferably less than 45, even more preferablyless than 40, even more preferably less than 35, even more preferablyless than 30, even more preferably less than 25, even more preferablyless than 20, even more preferably ≤15 amino acids long. Thus, linker Xcan be for example a linker as defined above for peptide linkers P orlinking sequence L. In a particularly preferred embodiment linker X mayfor example comprise the sequence GNGTSNGTS (SEQ ID NO:83), which allowsfor glycosylation of a polypeptide of the present invention and thusimproves stability of the overall polypeptide. The glycosylationresidues are then the Asn residues. Specific examples for sequences oflinker X may for example be AAAEFTRG (SEQ ID NO: 95), AAAGNGTSNGTSEFTRG(SEQ ID NO: 105), and GGSGNGTSNGTSG (SEQ ID NO: 106). The latter twoallow again for glycosylation of the polypeptide according to thepresent invention.

The structure of the polypeptide according to the present invention mayfor example comprise (from N- to C-Terminus; B: component B; X: peptidelinker X; A: a component A; P: peptide linker P):

-   -   B-X-A-P-A-P-A,        or may be    -   A-P-A-P-A-X-B.

Specific examples of polypeptides according to the present inventioncomprise as components A SEQ ID NO: 5 and as component B SEQ ID NO: 94,preferably with SEQ ID NOs: 48, 88 and/or 90 as peptide linkers P. SEQID NO: 102 and SEQ ID NO: 107 (SEQ ID NO: 107 corresponds to amino acids34-850 of SEQ ID NO: 102, i.e. does not include the leader sequence andthe FLAG tag) are preferred examples of the present invention. Furtherpreferred examples of the polypeptides according to the presentinvention, are sequences according to SEQ ID NOs: 125 and 126 comprisingan albumin binding domain (ABD), and sequences according to SEQ ID NOs:127-129 comprising a FAP-specific component B.

In a further aspect, the present invention relates to a polypeptidewhich comprises the sequence of SEQ ID NO: 96.

In a further aspect the present invention relates to a polypeptide whichcomprises:

-   -   a) at least three components A, each of which comprises the        sequence of a TNF homology domain (THD) of a TNF ligand family        member, or functional derivative thereof, and    -   b) a sequence comprising a glycosylation motif.

Glycosylation motifs comprise for instance nitrogen atoms in asparagineor arginine side-chains. Examples for glycosylation motifs are disclosedfor instance above in SEQ ID NOs: 83-91. Specific examples of suchpolypeptides are polypeptides comprising the sequence of SEQ ID NO: 97,or SEQ ID NO: 98.

Besides, the polypeptides according to the present invention shouldpreferably not comprise any endopeptidase recognition and/or cleavagesites, at least not within the structures B-X-A-P-A-P-A orA-P-A-P-A-X-B, respectively (presence of endopeptidase cleavage sites N-or C-terminal thereof will not affect the overall function of thepolypeptide according to the present invention and their presence isthus not critical). In other words, the polypeptide according to thepresent invention should preferably not comprise any endopeptidaserecognition and/or cleavage sites within the region comprising the atleast three components A and the at least one component B and the linkerin between. Presence of endopeptidease cleavage sites will significantlyreduce the half-life of a polypeptide according to the present inventionand may severely impact the efficacy of the polypeptide according to thepresent invention, because interaction of important domains isabolished. For example, separation of one component A from thepolypeptide according to the present invention via endopeptidasecleavage will prevent trimer formation. Likewise, if component B isseparated from the components A, any targeting effect is lost. This maybe prevented by removing/altering respective endopeptidase recognitionsites, by removing the endopeptidase cleavage sites and or by doingboth. In this context, it is particularly preferred, if the components Ain the polypeptide according to the present invention do not comprise aTACE cleavage site. TACE (TNF-alpha-converting enzyme, also termedADAM17) is a member of the ADAM protease family and represents theenzyme physiologically processing for example the initially membranebound TNF and others. TACE cleaves the membrane bound form whereby TNFis released (shed). Thus, a TNF based component A preferably lacks theAla76-Val77 cleavage site of TNF. Lacking the cleavage site implies thatthe cleavage site may be deleted or is altered e.g. by means ofsubstitution or insertion. Alternatively, or preferably in addition, theprotease binding site within the stalk region of a TNF ligand, e.g.amino acid 77-88 ofTNF-(Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Glu-Lys-Pro), may be altered(or preferably deleted) to avoid recognition of the polypeptide of thepresent invention by TACE.

Polypeptides according to the present invention may comprise furtherpolypeptide sequences and domains, which are however entirely optional.

One such optional element which may or may not be present in apolypeptide according to the present invention is the presence of one ormore albumin binding domains (ABDs) within a polypeptide according tothe present invention. The polypeptides according to the presentinvention may further comprise such an albumin binding domain inparticular with the purpose to prolong the plasma half-life of thepolypeptide of the present invention and thereby maintaintherapeutically effective plasma concentrations. Serum albumin possessesan extraordinary long plasma half-life in humans. The plasma half-lifeof human serum albumin is in the range of 19 days. Apart from IgG noother soluble serum protein is known to exhibit such long half-life. Thealbumin binding domain (ABD) may be any molecule with affinity foralbumin such as certain peptides, antibody fragments, alternativescaffolds, and small chemicals (for review see Kontermann BioDrugs 2009,23:93-109, incorporated herein by reference). Particularly preferredexamples of albumin binding domains in a polypeptide according to thepresent invention are selected for example from the group consisting of:albumin binding antibodies and albumin binding antibody derivatives,such as albumin binding Fab fragments, albumin binding scFv antibodies,and protein G of Streptococcus strain G148. Most preferred is the ABD ofprotein G of Streptococcus strain G148 comprising the sequenceQHDEAVDANSLAEAKVLANRE LDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP (SEQ ID NO:99) and non-immunogenic variants or derivatives thereof (e.g. asdescribed in Johnsson et al. Protein Eng. Des. Sel. 21 (2008) pp 515-527and Hopp et al. Protein Eng. Des. Sel. 23 (2010) pp 827-834, bothincorporated herein by reference). Alternatively, the polypeptideaccording to the present invention may simply be a fusion protein withalbumin moiety itself, or a fragment or derivative thereof. An exampleof such polypeptide is SEQ ID NO: 103.

In a particularly preferred embodiment, the polypeptide according to thepresent invention comprises an albumin binding domain as describedabove, for example an albumin binding domain according to SEQ ID NO: 99or a derivative thereof which is capable of binding albumin, such as asequence having at least 60% identity, preferably at least 70% identity,more preferably at least 80%, even more preferably at least 90% identityto SEQ ID NO: 99 over the entire length of SEQ ID NO: 99, or a fragmentof SEQ ID NO: 99 or of a derivative thereof capable of binding albumin,such as a fragment consisting of a continuous stretch of amino acidsrepresenting at least 40%, preferably at least 50%, preferably at least60%, more preferably at least 70%, even more preferably at least 80% ofthe full length sequence of SEQ ID NO: 99 or of a derivative thereof.

Preferably, the position of the ABD within the polypeptide according tothe present invention is selected such that it does not significantlyinterfere with the bioactivity of the polypeptide according to thepresent invention, e.g. of inducing apoptosis in target cells, such ascancer cells. It is particularly preferred that the optional ABD islocated between components A and component B of the polypeptideaccording to the present invention. For example, in a particularlypreferred embodiment, component B is located N-terminally to componentsA and the optional ABD is located between components A and component B,e.g. forming a fusion protein having the structure N-K-[componentB]-X-ABD-P-[components A]-X-C, wherein K is an optional V_(H) leadersequence, X and P are optional linkers as described herein which may ormay not comprise a glycosylation site, and N and C represent theN-terminal and the C-terminal end of the fusion protein, respectively.An example for such a polypeptide according to the present invention isgiven in SEQ ID NO: 125 (FIG. 25).

In another particularly preferred embodiment, the optional albuminbinding domain is located downstream of components A of the polypeptideaccording to the present invention. For example, in a particularlypreferred embodiment, component B is located N-terminally to componentsA and the optional ABD is located C-terminally to components A. Thus, ina particularly preferred embodiment, the polypeptide according to thepresent invention exhibits the structure N-K-[component B]-X-[componentsA]-P-ABD-X-C, wherein K is an optional V_(H) leader sequence, X and Pare optional linkers as described herein which may or may not comprise aglycosylation site, and N and C represent the N-terminal and theC-terminal end of the fusion protein, respectively. Preferably, ABD islocated at the C-terminal end of the polypeptide according to thepresent invention. An example of such a polypeptide according to thepresent invention is given in SEQ ID NO: 126 (FIG. 26).

Another optional element which may or may not be present in apolypeptide according to the present invention is a tag allowing forexample the detection and or purification of a polypeptide according tothe invention. Examples for such tags are for example a His-tag, aFLAG-tag (DYKDDDDK; SEQ ID NO: 100), a HA-tag, a STREP-tag, a myc-tag,GST. Preferably, such tag is positioned outside the region comprisingthe at least three components A and the at least one component B. If so,it is possible to position a protease cleavage site (such as a thrombincleavage site) adjacent to the tag, e.g. directly C-terminally of thetag. This will allow to remove the tag for example after purification.

Another optional but preferred element which may or may not be presentin a polypeptide according to the present invention is a leader orsignal peptide sequence such as the V_(H) leader sequenceMDWTWRVFCLLAVAPGAHS (SEQ ID NO: 101) or Igκ (METDTLLLWVLLLWVPGSTG; SEQID NO: 108). Such sequences may affect processing and targeting of thepolypeptide according to the present invention after translation ifproduced in matching cell systems. For example, the V_(H) leadersequence (if used for example in a mammalian) directs a polypeptideaccording to the present invention into the secretory pathway and thusallows for easier purification of the secreted product from culturesupernatants. As with the detection and purification tags mentionedabove, such leader or signal peptide sequence is positioned at the veryN-Terminus of the polypeptide according to the present invention, thusallowing a cotranslational translocation into the ER and physiologicalprocessing in suitable mammalian expression systems such as CHO cells.In this regard a person skilled in the art will understand that ifherein a polypeptide sequence is given with a leader sequence herein,then said polypeptide will for example after expression in a mammalianexpression no longer comprise said leader sequence. Certainly, any suchpolypeptide without leader sequence falls within the scope of thepresent invention. In particular, polypeptides comprising the sequenceof SEQ ID NOs: 96, 97, 98, 102, and/or 103 without the leader sequenceof SEQ ID NO: 101 are embodiments of the present invention. Likewise,polypeptides comprising the sequence of SEQ ID NOs: 96, 97, 98, 102,and/or 103 without the leader sequence of SEQ ID NO: 101 and without theFLAG tag sequence of SEQ ID NO: 100 are embodiments of the presentinvention.

Likewise a polypeptide according to the present invention may optionallyexhibit modifications. For instance, the polypeptide according to thepresent invention may be altered with regard to its hydrodynamic volume.The hydrodynamic volume of a protein can be increased by attachinghighly flexible, hydrophilic molecules such as polyethylene glycoland/or carbohydrates. PEGylation, i.e. the chemical coupling ofpolyethylene glycol (PEG) is frequently used in the art. PEG is composedof ethylene oxide units connected in a linear or branched configurationand of varying length. For example, one or several PEG chains of 5 to 40kDa may be conjugated to a polypeptide according to the presentinvention. However, PEGylation should preferably not be achieved inrandom manner because such approach may negatively impact thetrimerization or targeting properties of the polypeptides according tothe present invention. Preferably, the PEGylation sites are not withinthe region comprising the at least three components A and the at leastone component B. PEG moieties may for example be attached to thepolypeptide according to the present invention via cysteine residues.These cysteine residues are preferably positioned outside the regioncomprising the at least three components A and the at least onecomponent B. Several other techniques are also known in the art. PEGmimetics may certainly also be used to modify the polypeptides accordingto the present invention.

A further possible modification of the polypeptides according to thepresent invention is—as already indicated above—glycosylation.Glycosylation can positively influence the half-life and stability of apolypeptide according to the present invention. N- as well asO-glycosylation may be contemplated. The inventors of the presentinvention have for example shown that glycosylation is possible byintroducing a linker X which has the sequence GNGTSNGTS (SEQ ID NO: 83).Glycosylation sites at other sequence positions of the polypeptideaccording to the present invention are certainly also possible.Preferably, such glycosylation sites do not (significantly) impact thetargeting and the respective functional activity of the THD of the TNFligand family member within the polypeptide according to the presentinvention. Examples for such polypeptides are the polypeptides with thesequence of SEQ ID NO: 97, 98 and 103.

Other possible modifications of the polypeptides according to thepresent invention include for example HESylation (modification withhydroxyethyl starch) and modification with polysialic acid (PSA).

In general, the production of polypeptides is well-known in the art anda person skilled in the art can easily arrive at a polypeptide accordingof the present invention by means of routine methods (see for exampleManiatis, et al. (2001), Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press). In general, the production ofpolypeptides and proteins, respectively, is achieved by creating a DNAsequence encoding the same, followed by subsequent transformation of asuitable host with the respective DNA sequence and expression of themodified DNA sequence. Alternatively, the polypeptides according to thepresent invention may be chemically synthesized.

The present invention relates also to polypeptide complexes of thepolypeptides according to the present invention, e.g. homodimeric and/orhomotrimeric complexes of polypeptides according to the presentinvention. Thus, the polypeptide according to the present invention ispreferably capable of forming multimers, such as dimers, trimers,tetramers etc., preferably dimers. Preferably, component B is capable offorming multimers, such as dimers, trimers, tetramers etc. Thus, in aparticular preferred embodiment, component B is selected such thatmultimerization, such as dimerization, of the polypeptide according tothe present invention is possible. In a particularly preferredembodiment, the polypeptide according to the present invention exhibitsa multimeric form, such as a dimeric, trimeric, tetrameric etc. form,most preferably a dimeric form. Thus, preferably the polypeptideaccording to the present invention is multimeric, such as dimeric,trimeric or tetrameric, preferably dimeric. Accordingly, in a particularpreferred embodiment the polypeptide complex according to the presentinvention is dimeric. A dimeric polypeptide according to the presentinvention comprises at least 6 components A.

In a further aspect the present invention also relates to a nucleic acidencoding a polypeptide according to the present invention. The nucleicacid may be DNA or RNA or a hybrid thereof. Preferably, the nucleic acidalso comprises sequences allowing for the expression of the polypeptideaccording to the present invention in a suitable expression system. Thenucleic acid can be codon optimized for the respective expressionsystem.

In a further aspect the present invention also relates to a vectorcomprising a nucleic acid according to the present invention.Preferably, the vector provides for transcription and expression of thepolypeptide according to the present invention in a suitable host cellsystem.

In a further aspect the present invention also relates to a (host) cellcomprising a nucleic acid according to the present invention, a vectoraccording to the present invention, a polypeptide according to thepresent invention, or a polypeptide complex according to the presentinvention. If the host cell is a human host cell, it is an isolated hostcell outside the human body.

In a further aspect the present invention relates to a non-humanorganism comprising a nucleic acid according to the present invention, avector according to the present invention, a polypeptide according tothe present invention, a polypeptide complex according to the presentinvention or a host cell according to the present invention.

In a further aspect the present invention relates to nucleic acidaccording to the present invention, a vector according to the presentinvention, a polypeptide according to the present invention, apolypeptide complex according to the present invention and/or a hostcell according to the present invention in a method for treatment of thehuman or animal body by surgery or therapy and diagnostic methodspractised on the human or animal body. Preferably, the method oftreatment relates to the treatment of cancer, autoimmune or degenerativediseases.

In this context the present invention also relates to a pharmaceuticalcomposition comprising a nucleic acid according to the presentinvention, a vector according to the present invention, a polypeptideaccording to the present invention, a polypeptide complex according tothe present invention and/or a host cell according to the presentinvention and optionally a pharmaceutically acceptable carrier,adjuvant, and/or vehicle. A particularly preferred pharmaceuticalcomposition comprises a polypeptide comprising the sequence of SEQ IDNO: 102, 103, 107, 125, 126, 127, 128 and/or 129.

The pharmaceutical composition typically comprises a safe and effectiveamount of the compounds according to the invention (polypeptides,nucleic acids, vectors) as defined above. As used here, “safe andeffective amount” means an amount of the compounds as defined above,that is sufficient to significantly induce a positive modification of acondition to be treated, for example of cancer and/or a tumor. At thesame time, however, a “safe and effective amount” is preferably smallenough to avoid serious side-effects, that is to say to permit asensible relationship between advantage and risk. The determination ofthese limits typically lies within the scope of sensible medicaljudgment. A “safe and effective amount” of the compounds according tothe invention as defined above will vary in connection with theparticular condition to be treated and also with the age and physicalcondition of the patient to be treated, the severity of the condition,the duration of the treatment, the nature of the accompanying therapy,of the particular pharmaceutically acceptable carrier used, and similarfactors, within the knowledge and experience of the accompanying doctor.The medicament according to the invention can be used according to theinvention for human and also for veterinary medical purposes, as apharmaceutical composition.

The pharmaceutical composition of the present invention typicallycontains a pharmaceutically acceptable carrier. The expression“pharmaceutically acceptable carrier” as used herein preferably includesthe liquid or non-liquid basis of the inventive medicament. If theinventive medicament is provided in liquid form, the carrier willtypically be pyrogen-free water; isotonic saline or buffered (aqueous)solutions, e.g. phosphate, citrate etc. buffered solutions. Particularlyfor injection of the inventive medicament, a buffer, preferably anaqueous buffer, may be used, containing a sodium salt. The injectionbuffer may be hypertonic, isotonic or hypotonic with reference to thespecific reference medium, i.e. the buffer may have a higher, identicalor lower salt content with reference to the specific reference medium,wherein preferably such concentrations of the afore mentioned salts maybe used, which do not lead to damage of cells due to osmosis or otherconcentration effects. Reference media are e.g. in “in vivo” methodsoccurring liquids such as blood, lymph, cytosolic liquids, or other bodyliquids, or e.g. liquids, which may be used as reference media in “invitro” methods, such as common buffers or liquids. Such common buffersor liquids are known to a skilled person. Ringer-Lactate solution isparticularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well, which are suitable foradministration to a person. The term “compatible” as used here meansthat the constituents of the inventive medicament are capable of beingmixed with the compound according to the invention as defined above insuch a manner that no interaction occurs which would substantiallyreduce the pharmaceutical effectiveness of the inventive medicamentunder usual use conditions. Pharmaceutically acceptable carriers must,of course, have sufficiently high purity and sufficiently low toxicityto make them suitable for administration to a person to be treated. Someexamples of compounds which can be used as pharmaceutically acceptablecarriers or constituents thereof are sugars, such as, for example,lactose, glucose and sucrose; starches, such as, for example, cornstarch or potato starch; cellulose and its derivatives, such as, forexample, sodium carboxymethylcellulose, ethylcellulose, celluloseacetate; powdered tragacanth; malt; gelatin; tallow; solid glidants,such as, for example, stearic acid, magnesium stearate; calcium sulfate;vegetable oils, such as, for example, groundnut oil, cottonseed oil,sesame oil, olive oil, corn oil and oil from theobroma; polyols, suchas, for example, polypropylene glycol, glycerol, sorbitol, mannitol andpolyethylene glycol; alginic acid.

The choice of a pharmaceutically acceptable carrier is determined inprinciple by the manner in which the inventive medicaments areadministered. The inventive medicaments can be administered, forexample, systemically. Routes for administration include, for example,transdermale, inhalation, oral, parenteral, including subcutaneous orintravenous injections, topical and/or intranasal routes. The suitableamount of the inventive medicament to be administered can be determinedby routine experiments with animal models. Such models include, withoutimplying any limitation, rabbit, sheep, mouse, rat, dog and non-humanprimate models. Preferred unit dose forms for injection include sterilesolutions of water, physiological saline or mixtures thereof. The pH ofsuch solutions should be adjusted to about 7.4. Suitable carriers forinjection include hydrogels, devices for controlled or delayed release,polylactic acid and collagen matrices. Suitable pharmaceuticallyacceptable carriers for topical application include those which aresuitable for use in lotions, creams, gels and the like. If the inventivemedicament is to be administered perorally, tablets, capsules and thelike are the preferred unit dose form. The pharmaceutically acceptablecarriers for the preparation of unit dose forms which can be used fororal administration are well known in the prior art. The choice thereofwill depend on secondary considerations such as taste, costs andstorability, which are not critical for the purposes of the presentinvention, and can be made without difficulty by a person skilled in theart.

All references cited herein are incorporated herein in their entirety.

In the following a brief description of the appended figures will begiven. The figures are intended to illustrate the present invention inmore detail. However, they are not intended to limit the subject matterof the invention to any extent.

FIG. 1: Schematic illustration of exemplary polypeptides according tothe present invention:

A) K=V_(H) leader (e.g. SEQ ID NO: 101);

-   -   F=tag (i.e. FLAG tag; see for instance SEQ ID NO: 100);    -   L=linker sequence L, e.g. 15 aa long (e.g. (GGGGS)₃; SEQ ID NO:        52);    -   X=peptide linker X (see for instance SEQ ID NO: 95);    -   P=peptide linker P (e.g. SEQ ID NO: 48);    -   A=component A (e.g. TRAIL aa residues 95-281, SEQ ID NO: 5).    -   An example for such polypeptide is SEQ ID NO: 96 (FIG. 20).

B) K=V_(H) leader (e.g. SEQ ID NO: 101);

-   -   F=tag (i.e. FLAG tag; see for instance SEQ ID NO: 100);    -   L=linker sequence L, ≤12 aa long (e.g. (GGGGS); SEQ ID NO:50);    -   X=peptide linker X (see for instance SEQ ID NO: 95);    -   P=peptide linker P (e.g. SEQ ID NO: 48);    -   A=component A (e.g. TRAIL aa residues 95-281; SEQ ID NO: 5).    -   An example for such polypeptide is SEQ ID NO: 102 (FIG. 21).

C) K=V_(H) leader (e.g. SEQ ID NO: 101);

-   -   F=tag (i.e. FLAG tag; see for instance SEQ ID NO: 100);    -   L=linker sequence L, e.g. 15 aa long (e.g. (GGGGS)₃; SEQ ID NO:        52);    -   X=peptide linker X including glycosylation site (see for        instance SEQ ID NO: 105);    -   P=peptide linker P (e.g. SEQ ID NO: 48);    -   A=component A (e.g. TRAIL aa residues 95-281; SEQ ID NO: 5).    -   An example for such polypeptide is SEQ ID NO: 97 (FIG. 22).

D) K=V_(H) leader (e.g. SEQ ID NO: 101);

-   -   F=tag (i.e. FLAG tag; see for instance SEQ ID NO: 100);    -   L=linker sequence L, e.g. 15 aa long (e.g. (GGGGS)₃; SEQ ID NO:        52);    -   X=peptide linker X including glycosylation site (see for        instance SEQ ID NO: 105);    -   P=peptide linker P including glycosylation site (e.g. P₁: (SEQ        ID NO: 90); P₂: SEQ ID NO: 88);    -   A=component A (e.g. TRAIL aa residues 95-281; SEQ ID NO: 5).    -   An example for such polypeptide is SEQ ID NO: 98 (FIG. 23).

E) K=V_(H) leader (e.g. SEQ ID NO: 101);

-   -   ABD=Albumin binding domain (see for instance SEQ ID NO: 99);    -   Q=Linker sequence (e.g. GGSGGGGSGG; SEQ ID NO: 71);    -   L=linker sequence L, ≤12 aa long (e.g. (GGGGS); SEQ ID NO: 50);    -   X=peptide linker X including glycosylation site (see for        instance SEQ ID NO: 106);    -   P=peptide linker P with (e.g. SEQ ID NO: 88) or without        glycosylation site (e.g. SEQ ID NO 48: GGGSGGGS);    -   A=component A (e.g. TRAIL aa residues 95-281, SEQ ID NO: 5).    -   An example for such polypeptide is SEQ ID NO: 103 (FIG. 24).

F) K=V_(H) leader (e.g. SEQ ID NO: 101);

-   -   F=tag (i.e. FLAG tag; see for instance SEQ ID NO: 100);    -   L=linker sequence L, ≤12 aa long (e.g. (GGGGS); SEQ ID NO: 50);    -   Q=Linker sequence (e.g. GGS; SEQ ID NO: 55);    -   ABD=Albumin binding domain (see for instance SEQ ID NO: 99);    -   X=peptide linker X including glycosylation site (see for        instance SEQ ID NO: 105);    -   P=peptide linker P (e.g. GGGSGGGS; SEQ ID NO: 48);    -   A=component A (e.g. TRAIL aa residues 95-281, SEQ ID NO: 5).    -   An example for such polypeptide is SEQ ID NO: 125 (FIG. 25).

G) K=V_(H) leader (e.g. SEQ ID NO: 101);

-   -   F=tag (i.e. FLAG tag; see for instance SEQ ID NO: 100);    -   L=linker sequence L, ≤12 aa long (e.g. (GGGGS); SEQ ID NO: 50);    -   X=peptide linker X including glycosylation site (see for        instance SEQ ID NO: 105);    -   P₁=peptide linker P₁ (e.g. GGGSGGGS; SEQ ID NO: 48);    -   P₂=peptide linker P₂ (e.g. GGSGG; SEQ ID NO: 61);    -   A=component A (e.g. TRAIL aa residues 95-281, SEQ ID NO: 5);    -   ABD=Albumin binding domain (see for instance SEQ ID NO: 99).    -   An example for such polypeptide is SEQ ID NO: 126 (FIG. 26).

FIG. 2: Purified polypeptides of SEQ ID NO: 104 (lane 1), SEQ ID NO: 96(lane 2), SEQ ID NO: 102 (lane 3) and of glycosylated SEQ ID NO: 97(lane 4) were analyzed by SDS-PAGE (reducing conditions) followed bysilver staining (upper, left, 1 μg protein per lane) or Western blotting(lower, 250 ng protein per lane) using monoclonal anti-TRAIL oranti-FLAG antibodies in combination with alkaline phosphatase-conjugatedsecondary antibody. The glycosylated polypeptide of SEQ ID NO: 97 wastreated with N-glycosidase and analysed by SDS-PAGE and Coomassiestaining (upper, right). SEQ ID NO: 104 is a single chain TRAILpolypeptide with three copies of TRAIL 95-281 (SEQ ID NO: 5) linked bytwo glycine linkers and comprising an N-terminal Flag-tag. However, thepolypeptide does not comprise any region B as specified herein.

FIG. 3 Purified polypeptides of SEQ ID NO: 104 (upper left), SEQ ID NO:96 (lower left), SEQ ID NO: 102 (upper right) and of glycosylated SEQ IDNO: 97 (lower right) were separated by size exclusion chromatography ona BioSuite 250 column. The retention times of the molecular weightstandards thyroglobulin (669 kDa), β-amylase (200 kDa), bovine serumalbumin (67 kDa) and carbonic anhydrase (29 kDa) are indicated by dottedlines.

FIG. 4 Flow cytometric analysis of expression of EGF receptor andproapoptotic TRAIL receptors DR4 and DR5 in Jurkat, Huh-7 and HepG2 celllines.

FIG. 5 Flow cytometric analysis: (A) Blocking of the binding ofCetuximab to target-negative (HepG2) and target-positive cells (Huh-7)by an excess of SEQ ID NO: 102; (B) Binding of SEQ ID NO: 102 and SEQ IDNO: 96 to HepG2 and Huh-7 cells. “scFv” represents an anti-EGFR specificantibody fragment used for competition.

FIG. 6 Target-independent induction of cell death: (A) EGFR low/negativeHepG2 cells were sensitized with 500 ng/ml Bortezomib and treated withserial dilutions of SEQ ID NO: 102 (open squares), KillerTRAIL™(inverted filled triangle), SEQ ID NO: 96 (open circles) and SEQ IDNO:104. Cell viability was determined using crystal violet staining.Results from four independent experiments are shown (mean±S.E.M.). (B)Jurkat cells (1×10⁵/well) were used for a similar experiment asdescribed in (A). Jurkat cells were not sensitized. Results from threeindependent experiments are shown (mean±S.E.M.). Viability of Jurkatcells was determined using the MTT assay.

FIG. 7. EGFR-directed induction of cell death in EGFR+ Huh-7hepatocellular carcinoma cells sensitized with 250 ng/ml Bortezomib andtreated in duplicates with serial dilutions of SEQ ID NO: 102 (opensquares), SEQ ID NO: 96 (open circles), SEQ ID NO: 97 (open diamond),and SEQ ID NO: 104 as control (left side of panel). For quantificationof the targeting effect, cells were additionally preincubated with anexcess of EGFR-specific antibody Cetuximab (70 nM) before adding thetest polypeptides (graphs of SEQ ID NO: 102 (middle panel) and SEQ IDNO: 96 (right panel). For easy comparison, dose response curves of therespective reagents in absence of cetuximab from the left panel wereplotted again in these two panels. Results from four independentexperiments are shown (mean±S.E.M.).

FIG. 8: EGFR-directed induction of cell death in Huh-7 hepatocellularcarcinoma cells as in FIG. 7 with the exception that constantconcentrations of the test proteins were used for preincubation (30min), followed by addition of serial dilutions of Bortezomib. Control:Bortezomib alone. Results from three independent experiments are shown(mean±S.E.M.).

FIG. 9: EGFR specific TRAIL fusion proteins lack hepatotoxic activity.Groups of 3 CD1 mice were treated intraperitonally with indicated fusionproteins or control reagents: negative control: PBS; positive control:aggregated FasL (CD95L) fusion protein. (A) Plasma samples were preparedafter 4 h and 24 h and the activity of alanine aminotransferase (ALT)was assayed using an enzymatic assay (Bioo Scientific, Austin, Tex.).Dashed line indicates upper normal level of ALT (physiologic range inadult human: 35-50 U/L). (B) Mice were sacrificed after 24 h except forpositive control (animals treated with an aggregated FasL (CD95L) fusionprotein show phenotypic signs of severe systemic toxicity after 2-4 hrsand die after ˜5 hrs, samples were taken after 4 hrs) and liver biopsieswere taken for determination of caspase-3 activity using a specificAMC-coupled peptide substrate (Enzo Lifesciences, Lörrach, Germany).

FIG. 10: EGFR-directed induction of cell death by single-chain TRAILfusions on NCI-H460 cells. NCI-H460 non-small lung cancer cells(3×10⁴/well) were seeded in 96-well plates and cultivated for 24 h.Then, cells were sensitized with 2.5 μg/ml cycloheximide and treated induplicates with serial dilutions of the indicated fusion proteins. After16 h, cell viability was determined using crystal violet staining.Results from two independent experiments are shown (mean±S.E.M.). EC₅₀values were 1.2±0.08×10⁻¹² M for SEQ ID NO: 96, 3.4±0.28×10⁻¹³ M for SEQID NO: 102 and 5.8±1.5×10⁻¹² M for SEQ ID NO: 98.

FIG. 11: Biochemical characterization of N-glycosylated polypeptideaccording to the present invention (SEQ ID NO: 98). (A) SEQ ID NO: 98was treated with N-glycosidase and analysed by SDS-PAGE and Coomassiestaining. (B) SEQ ID NO: 98 and SEQ ID NO: 96 were separated by sizeexclusion chromatography on a BioSuite 450 column (Waters). Theretention times of the molecular weight standards apoferritin (443 kDa),β-amylase (200 kDa), bovine serum albumin (67 kDa) and carbonicanhydrase (29 kDa) were indicated by dotted lines.

FIG. 12: Receptor interaction of EGFR-specific TRAIL fusion proteins.(A) Dose response relationship of TRAIL fusion protein binding to EGFR⁺NCI-H460 cells by indirect immunofluorescence flow cytometry to revealconcentration of half maximum binding (EC₅₀) (mean SEM, n=4). (B)Colo205 and Huh-7 cells were serum-starved overnight and then incubatedwith 2 nM of SEQ ID NO: 102, Cetuximab, and PBS for control,respectively. After 10 min, 50 ng/ml EGF were added and cells wereincubated for additional 20 min followed by cell lysis. EGF receptorswere immunoprecipitated using a specific mouse monoclonal antibody andsubjected to SDS-PAGE followed by immunoblotting with phosphotyrosineantibody (anti-pTyr). Total amounts of EGFR were determined by reprobingthe membrane with EGFR-specific rabbit polyclonal antibody (anti-EGFR).

FIG. 13: Caspase dependence of cell death and impact of the component Bof SEQ ID NO: 102. (A) Colo205 cells (left) and Huh-7 cells (right) weresensitized with 25 ng/ml and 250 ng/ml bortezomib, respectively, andtreated with different concentrations of SEQ ID NO: 102 with or withoutthe presence of pan-caspase inhibitor zVADfmk or caspase-3 inhibitorzDEVDfmk (both inhibitors: 20 μM for Colo205 and 10 μM for Huh-7). After16 h, cell viability was determined using MTT staining (Colo205) orcrystal violet staining (Huh-7) and data were normalized usingbortezomib-treated cells as control (mean SEM, n=3). (B) 1×10⁴ Colo205cells per well were grown in 96-well plates using medium with 0.1% FCS.Upon stimulation with 50 ng/ml EGF and sensitization with 10 ng/mlbortezomib, cells were incubated with equimolar concentrations of SEQ IDNO: 102 (open squares), SEQ ID NO: 102+anti-TRAIL mAb 2E5 (filledsquares) or Cetuximab (circles) for four days and cell number wasassayed by the MTT method using bortezomib/EGF-treated cells as controlfor normalization (mean SEM, n=2). (C) 1×10⁴ Huh-7 cells per well weregrown in 96-well plates and treated with 20 ng/ml bortezomib or with acombination of bortezomib and 10 μM zVADfmk. Then, cells were incubatedwith equimolar concentrations of SEQ ID NO: 102 (open squares), SEQ IDNO: 102+zVADfmk (filled squares), Cetuximab (open circles) orCetuximab+zVADfmk (filled circles) for three days and cell viability wasassayed by the MTT method using bortezomib-treated cells andbortezomib/zVADfmk-treated cells, respectively, as control fornormalization (mean SEM, n=3).

FIG. 14: In vitro tolerance of TRAIL fusion proteins to primary tissues.(A) Relative caspase activity (fold increase compared to untreated) inprimary human hepatocytes (PHH, mean SEM, n=5) or Huh-7 hepatocarcinomacells (mean SEM, n=7) after incubation with 1.1 nM SEQ ID NO: 102 inpresence or without 500 ng/ml bortezomib. Asterisks indicate statisticalsignificance. (B) Cleavage of caspase-3 in PHH (left) and Huh-7 cells(right) after incubation with 500 ng/ml bortezomib, 1.1 nM SEQ ID NO:102 or both was analyzed by immunoblotting.

FIG. 15: Antitumor activity of TRAIL fusion proteins in a Colo205xenograft tumor model. (A) Tumor volume as a function of time after i.p.application of PBS (open diamonds), bortezomib (filled triangles), SEQID NO: 104 (lacking component B)+bortezomib (open triangles), SEQ ID NO:97 (L>12 amino acids)+bortezomib (circles), SEQ ID NO: 102 (L<12 aminoacids)+bortezomib (filled diamonds) or SEQ ID NO: 102 only (squares).Arrows, protein application; asterisks, bortezomib application; symbols,mean of tumor volumes 95% confidence interval (CI), n=12tumors/treatment group. (B) Individual tumor volumes at day 14. Bars,mean of tumor volumes 95% CI.

FIG. 16: Coomassie-stained SDS-PAGE of affinity-purified SEQ ID NO: 102(lane 1), SEQ ID NO: 125 (lane 2) and SEQ ID NO: 126 (lane 3). 5 μg ofthe proteins were loaded under reducing conditions.

FIG. 17: Albumin binding of SEQ ID NO: 125 and SEQ ID NO: 126. Bothproteins were incubated for 1 h at RT with equimolar concentrations ofhuman serum albumin (HSA) or mouse serum albumin (MSA) and subsequentlyseparated by size exclusion chromatography. Thyroglobulin (669 kDa),apoferritin (443 kDa), -amylase (200 kDa), bovine serum albumin (67kDa), carbonic anhydrase (29 kDa) and FLAG peptide (1 kDa) were used asstandard proteins/peptides.

FIG. 18: Bioactivity of SEQ ID NO: 125 and SEQ ID NO: 126 in vitro.Huh-7 hepatocarcinoma cells were sensitized with bortezomib (250 ng/ml)and treated with serial dilutions of SEQ ID NO: 102, SEQ ID NO: 125(left panel) and SEQ ID NO: 126 (right panel) in triplicates. After 16h, cell viability was determined using crystal violet staining. Forquantification of the targeting effect, cells were preincubated with anexcess of Cetuximab (70 nM) before adding SEQ ID NO: 102, SEQ ID NO: 125and SEQ ID NO: 126. SEQ ID NO: 125 and SEQ ID NO: 126 were additionallyincubated in presence of 100 μg/ml HSA. The values for SEQ ID NO: 102were plotted in both panels for comparison.

FIG. 19: Pharmacokinetics of SEQ ID NO: 102 (A) and SEQ ID NO: 125 (B).25 μg of protein were injected i.v. in CD1 mice and serum samples weretaken at the depicted time points followed by detection of scTRAILmolecules via ELISA.

FIG. 20A-20C: SEQUENCE OF SEQ ID NO: 96.

FIG. 21A-21C: SEQUENCE OF SEQ ID NO: 102.

FIG. 22A-22C: SEQUENCE OF SEQ ID NO: 97.

FIG. 23A-23C: SEQUENCE OF SEQ ID NO: 98.

FIG. 24A-24C: SEQUENCE OF SEQ ID NO: 103.

FIG. 25A-25C: SEQUENCE OF SEQ ID NO: 125.

FIG. 26A-26C: SEQUENCE OF SEQ ID NO: 126.

FIG. 27: Sequence of SEQ ID NO: 127.

FIG. 28: Sequence of SEQ ID NO: 128.

FIG. 29: Sequence of SEQ ID NO: 129.

EXAMPLES

In the following, general examples illustrating various embodiments andaspects of the invention are presented. However, the present inventionshall not be limited in scope by the specific embodiments describedherein. Indeed, various modifications of the invention in addition tothose described herein will become readily apparent to those skilled inthe art from the foregoing description, accompanying figures and theexamples below. All such modifications fall within the scope of theappended claims.

Example 1: Biochemical Analysis

1. Polypeptide Production

1.1 Principle

Three human TRAIL domains encompassing aa residues 95-281 (TRAIL) (SEQID NO: 5) were fused with (GGGS)₂ peptide linkers P (SEQ ID NO: 48)yielding so called single-chain TRAIL (scTRAIL) (SEQ ID NO: 104).EGFR-specific antibody fragments consisting of V_(H) (SEQ ID NO: 93) andV_(L) (SEQ ID NO: 92) were fused N-terminally to scTRAIL (SEQ ID NO:104). (GGGGS)₃ (SEQ ID NO: 52) or GGGGS (SEQ ID NO: 50) peptide linkersbetween V_(H) (SEQ ID NO: 93) and V_(L)(SEQ ID NO: 92) were chosen toobtain a polypeptide according to the present invention (SEQ ID NOs: 96and 102). A V_(H) leader (K) (SEQ ID NO: 101) and a FLAG tag (F) (SEQ IDNO: 100) were placed in front of the antibody region. For a glycosylatedpolypeptide according to the present invention, a linker with twoN-glycosylation sites (GNGTSNGTS) (SEQ ID NO: 83) was placed betweenV_(L) (SEQ ID NO: 92) and scTRAIL (SEQ ID NO: 104).

1.2 Plasmids and Cell Lines

An pIRESpuro-scTRAIL expression construct for human scTRAIL (SEQ ID NO:104) was obtained by EcoRI/NotI cloning of a synthesized sequence codingfor three TRAIL components (aa residues 95-281) connected by sequencesencoding (GGGS)₂ linker motifs into a construct described previously(Schneider et al, 2010, Cell Death. Disease. 2010). For the generationof the EGFR-specific V_(H)-V_(L)-scTRAIL expression construct, asynthesized coding sequence of humanized V_(H) and V_(L) sequences(huC225) was amplified using the oligonucleotides CGAGGTGCAGCTGGTCGAG(SEQ ID NO: 109) and TGCGGCCGCTCTCTTGATTTC (SEQ ID NO: 110). Next, thistemplate was annealed with the oligonucleotideATATATCTCGAGGCCAGCGACTACAAAGACGATGACGATAAAGGAGCCGAGGTGCAGCTGG TCGAG (SEQID NO: 111) to insert an XhoI site and a FLAG tag coding sequence. Afterstrand elongation, the whole sequence was amplified by theoligonucleotides ATATATCTCGAGGCCAGCGAC (SEQ ID NO: 112) andATATGAATTCTGCGGCCG CTCTCTTGATTTC (SEQ ID NO: 113). The PCR product wasthen cloned via XhoI/EcoRI into pCR3 (Invitrogen), carrying an V_(H)leader. The scTRAIL coding sequence of this construct was then insertedvia EcoRI/XbaI sites. The EGFR-specific construct for SEQ ID NO: 102 wasderived from pCR3-VH-VL-scTRAIL by shortening linker L from (GGGGS)₃ toGGGGS. Therefore, two PCR products were generated using theoligonucleotide

(1) (SEQ ID NO: 114) CCCACAGCCTCGAGGCCAG and (2) (SEQ ID NO: 115)GAGCCGCCACCGCCACTAGvas well as

(3) (SEQ ID NO: 116) CTAGTGGCGGTGGCGGCTCTGATATTCAGCTGA CCCAGTCC and (4)(SEQ ID NO: 117) TGAATTCTGCGGCCGCTCTC.After annealing of the products at the underlined regions and strandelongation, the whole sequence was amplified by the oligonucleotides (1)and (4) followed by XhoI/NotI cloning into pCR3-V_(H)-V_(L)-scTRAIL. Aglycosylated variant of SEQ ID NO: 96 was generated by two PCRamplifications of the huC225 VH-VL coding sequence in pCR3-VH-VL-scTRAILusing the oligonucleotides (1) CCCACAGCCTCGAGGCCAG (SEQ ID NO: 118) andCCCGTTGCTGGTGCCGTTGCCTGCG GCCGCTCTCTTG (SEQ ID NO: 119), respectively(1) and ATATGAATTCGGATGTCCCGTT GCTGGTGCCGTTG (SEQ ID NO: 120), followedby XhoI/EcoRI cloning in pCR3-VH-VL-scTRAIL. The construct forexpression of SEQ ID NO: 98 was generated by two sequential PCRamplifications of the TRAIL coding sequence using the oligonucleotidesGCACATCCAATGGGACCAGCGGAACCTCCGAAGAGACTATCTC (SEQ ID NO: 121) andCCCGTTGCTGGTTCCATTACCAGATCCGCCCCCTCC (SEQ ID NO: 122), respectivelyATATATGGATCCGGCAACGGCACATCCAATGGGACCAG (SEQ ID NO: 123) andATATATGGATCCGGTCCCGTTGCTGGTTCCATTAC (SEQ ID NO: 124), followed by BamHIcloning into the expression construct for SEQ ID NO: 97.

HEK293, HepG2, NCI-H460, Colo205 and Jurkat, cells were obtained fromthe American Type Culture Collection (Manassas, Va.). Cells werecultured in RPMI 1640 medium (Invitrogen, Karlsruhe, Germany)supplemented with 5% fetal calf serum (FCS, HyClone), respectively 10%FCS for HepG2. Huh-7D12 liver carcinoma cells were obtained from HeikeBantel, Hannover Medical School, Hannover, Germany and were cultured inDMEM (Invitrogen, Karlsruhe, Germany) supplemented with 10% FCS.

1.3 Production and Purification of Recombinant Proteins

The TRAIL fusion proteins of SEQ ID NOs: 96, 97, 98, 102, 125, and 126were produced in HEK293 cells after stable transfection with thecorresponding expression plasmids using Lipofectamine 2000 (Invitrogen)and generation of a pool of stably expressing clones. For proteinproduction, stable clones were expanded and grown in RPMI 1640, 5% FCS,to 90% confluency and subsequently cultured in serum-free Optimem(Invitrogen) supplemented with 50 μM ZnCl₂, replacing media two timesevery 3 days. The supernatants were pooled and recombinant proteins werepurified first by IMAC using Ni-NTA-Agarose (Qiagen, Hilden, Germany).After elution with 100 mM imidazol and dialysis against PBS, theproteins were further purified by affinity chromatography usinganti-FLAG mAb M2 agarose (Sigma-Aldrich, Steinheim, Germany). The boundproteins were eluted with 100 μg/ml FLAG peptide (peptides&elephants,Potsdam, Germany) and dialysed against PBS. scTRAIL and SEQ ID NOs: 97and 98 were purified in a single M2 agarose affinity chromatographystep. After concentration of purified proteins using Vivaspincentrifugal concentrators with 50 or 10 kDa MWCO (Sartorius Stedim,Aubagne, France), the protein concentration was measured with aspectrophotometer (NanoDrop products, Wilmington, Del.) and aliquotswere stored at −80° C.

1.4 SDS-PAGE and Western Blot Analysis

Purified polypeptides of SEQ ID NO: 96, SEQ ID NO: 102, SEQ ID NO: 104,125, 126 and of glycosylated SEQ ID NO: 97 were analyzed by SDS-PAGE(reducing conditions) followed by silver staining (1 μg protein perlane), Coomassie staining or Western blotting (250 ng protein per lane)using monoclonal anti-TRAIL (MAB687, R&D Systems, Wiesbaden, Germany) oranti-FLAG antibodies (M2, Sigma-Aldrich) in combination with alkalinephosphatase-conjugated secondary antibody (Sigma-Aldrich). Theglycosylated polypeptide of SEQ ID NO: 97 was treated with N-glycosidaseand analysed by SDS-PAGE and Coomassie staining. For deglycosylation,protein (5 μg) was denatured in the presence of SDS and DTT prior toaddition of Nonidet P-40 and 500 units of PNGaseF (New England Biolabs,Frankfurt a. M., Germany) according to the supplier's instructions.After 1 h incubation at 37° C., samples were subjected to SDS-PAGE. ForWestern blotting, an anti-TRAIL antibody MAB687 (R&D Systems, Wiesbaden,Germany) and anti-FLAG M2 mAb (Sigma-Aldrich) were used, followed by ananti-mouse alkaline phosphatase-coupled secondary antibody(Sigma-Aldrich) for detection.

The results of the SDS PAGE/Western Blot analysis verified the increasein molecular mass of SEQ ID NOs: 96, 97 and 102 vs. SEQ ID NO: 104. Theincrease of molecular mass of SEQ ID NOs: 125 and 126 vs. SEQ ID NO: 102has been verified by SDS page and Coomassie staining. SEQ ID NO: 97showed reduced migration in SDS PAGE, which is conform with effectiveglycosylation of the protein. This was confirmed by PNGaseF treatment ofthe fusion protein, which removes carbohydrate side chains inglycoproteins, resulting in a shift of the specific band towards themass of the non-glycosylated form. Furthermore, the assays confirmed thepresence of TRAIL as well as of the FLAG-tag in all 4 polypeptides.

1.5 Size Exclusion Chromatography and Albumin Binding Assay

Purified polypeptides of SEQ ID NO: 96, SEQ ID NO: 102, SEQ ID NO: 104and of glycosylated SEQ ID NO: 97 were separated by size exclusionchromatography on a BioSuite 250 HR SEC (300×7.8) column (Waters,Millipore Corp., Milford, Mass.) equilibrated in PBS and eluted at aflow rate of 0.5 ml/min.

Albumin binding to the polypeptides of SEQ ID NOs: 125 and 126 has beenverified by incubating the polypeptides with human serum albumin ormouse serum albumin and determining protein-protein interaction by sizeexclusion chromatography as described above.

1.6 Immunoprecipitation and Protein Analysis

For immunoprecipitations, cells were lysed on ice in RIPA buffer (50 mMTris, pH 7.5, 150 mM NaCl, 10 mM sodium fluoride, 20 mMglycerophosphate, 1 mM EDTA, 1% NP40, 1 mM sodium orthovanadate, 0.5 mMphenylmethylsulfonyl fluoride, 0.1% SDS, 0.25% sodium deoxycholate) withComplete protease inhibitor (Roche Diagnostics, Mannheim, Germany) andlysates were clarified by centrifugation (16 000 g, 10 min, 4° C.). 1.5mg lysate protein was incubated with 1.5 μg mouse anti-EGFR Ab-13 mAb(Neomarkers, Fremont, Calif., USA) under gentle shaking at 4° C.overnight. Immune complexes were captured with protein G sepharose (KPL,Gaithersburg, Md., USA) and washed three times with RIPA buffer.Proteins were analyzed by SDS-PAGE and Western blotting using mouseanti-phosphotyrosine P-Tyr-100 mAb (Cell Signaling Technology, Danvers,Mass., USA) and rabbit anti-EGFR 1005 antibody (Santa CruzBiotechnology, Santa Cruz, Calif., USA) followed by HRP-conjugatedsecondary antibodies. ECL (Pierce Biotechnology, Rockford, Ill., USA)was used for visualization.

Caspases were detected by immunoblotting using a rabbit polyclonalantibody against cleaved caspase-3 (Cell Signaling Technology). GAPDH asinternal control was detected with a rabbit polyclonal antibody (CellSignaling Technology). HRP-conjugated secondary antibodies (ZymedLaboratories, San Fransisco, Calif., USA) and ECL were used forvisualization.

Example 2: Flow Cytometry, Cell Death Assay, ALT and Caspase Activities

2.1 Flow Cytometry

5×10⁵ cells were suspended in PBA buffer (PBS, 0.025% BSA, 0.02% sodiumazide) and incubated for 1 h at 4° C. with the indicated scTRAIL fusionproteins (2 μg/ml). After washing the cells three times with PBA buffer,bound fusion proteins were detected by anti-human TRAIL mAb MAB687 (2.5μg/ml, R&D Systems) and fluorescein isothiocyanate-labelled rabbitanti-mouse IgG Ab (1:200, Sigma-Aldrich), followed by three washingsteps with PBA each. For blocking of scTRAIL fusion protein binding toEGFRs (see FIG. 5B), a divalent variant of huC225 (huC225Cys, 50 μg/ml,kindly provided by Celonic GmbH, Jülich, Germany) was added 30 minbefore addition of SEQ ID NO: 96 and SEQ ID NO: 102, respectively.Expression of TRAIL receptors was detected by anti-TRAIL R1 mAb MAB347and anti-TRAIL R2 mAb MAB6311 (4 μg/ml each, R&D Systems) in conjunctionwith anti-mouse IgG-FITC. EGFR expression was detected by aphycoerythrin-labelled anti-human EGFR mAb sc-101 (4 μg/ml, Santa CruzBiotech., Santa Cruz, Calif.) (see FIG. 4). For binding inhibition,purified SEQ ID NO: 102 (50 μg/ml) was added 30 min prior addition ofAlexa Fluor 488-coupled mAb Cetuximab (1 μg/ml) (see FIG. 5A).

The assays determined the expression levels of EGF receptor andproapoptotic TRAIL receptors DR4 and DR5 in Huh-7 and Hep2Ghepatocellular carcinoma cell lines and the T cell leukemia line Jurkat,revealing Huh7 as EGFR+, DR4+, DR5 low; HepG2 as EGFR low/negative,DR4+, DR5+; and Jurkat as EGFR negative, DR4 negative, DR5 low (see FIG.4). Furthermore, blocking of the binding of anti EGFR mab Cetuximab toEGFR+ cells (Huh-7) by an excess of EGFR receptor specific TRAIL protein(SEQ ID NO: 102) revealed functional expression of the EGFR specificV_(H)-V_(L) domain within the fusion protein of SEQ ID NO: 102 (FIG. 5A,right panel); as expected, the marginal Cetuximab staining of EGFRlow/negative HepG2 cells could not be further reduced by an excess ofthe fusion protein of SEQ ID NO: 102, and likely reflect nonspecificbackground staining of the reagent. Likewise, binding of the EGFRtargeting fusion proteins SEQ ID NO: 102 and SEQ ID NO: 96 to EGFR+Huh-7 cells was partially blockable by an excess of an anti-EGFRspecific antibody fragment, whereby the remaining signal could beattributed to specific binding of the fusion proteins via the TRAILdomain to their cognate TRAIL receptors. In this experimental setting,binding of fusion proteins was clearly discernable for DR4+DR5+HepG2cells, too, with little blocking of the signal by addition of ananti-EGFR specific antibody fragment due to low expression of EGFRs ator below the detection level in these cells.

2.2 Cell Death Assays

Huh-7 (3×10⁴), HepG2 (3×10⁴), Colo205 (5×10⁴ per well) or Jurkat cells(1×10⁵) were grown in 100 μl culture medium in 96-well plates for 24 h,followed by treatment with the indicated concentrations of SEQ ID NOs:96, 102, 125, 126 and 104 or ‘KillerTRAIL’ (Axxora Deutschland GmbH,Lörrach, Germany) in triplicates (see FIGS. 6-8 and 18). As a positivecontrol, cells were killed with 0.25% Triton X-100. Cell death assayswith Huh-7 and HepG2 cells were performed in the absence (FIG. 6, Jurkatcells) or presence of Bortezomib (FIG. 7, Huh-7: 250 ng/ml, FIG. 6,HepG2: 500 ng/ml, FIG. 18, Huh-7: 250 ng/ml), Selleck Chemicals,Houston, Tex.). Bortezomib was added 30 min prior incubation with theproapoptotic ligands to sensitize cells for the induction of cell death(FIGS. 6, 7). Alternatively, cells were preincubated for 30 min with theindicated concentrations of TRAIL fusion proteins followed by additionof serial dilutions of Bortezomib (FIG. 8). TRAIL only treated cells areshown in each panel for the applied TRAIL concentration (Bortezomib 0ng/ml) (FIG. 8). After 16 h incubation, cell viability was determinedeither by crystal violet staining (Huh-7, HepG2) or the MTT method(Jurkat) (Wuest et al., 2002). In the latter case a lysis bufferconsisting of 15% SDS in DMF/H₂O (1:1), pH 4.5 (with 80% acetic acid)was used. To demonstrate target antigen-dependent induction of celldeath, cells were preincubated for 30 min with competing Cetuximab mAb(10 μg/ml, Merck, Darmstadt, Germany) (FIG. 18) or alternatively EGFRspecific huC225Cys (10 μg/ml) (FIG. 7).

2.3 Alanine Aminotransaminase (ALT) and Caspase Activities

Groups of three CD1 mice (Janvier, Le Genest-St-Isle, France) weretreated i.p. with 1 nmol of fusion proteins according to SEQ ID NO: 102and SEQ ID NO: 97, 0.1 nmol FasL fusion protein (positive control) andPBS (negative control), respectively. Blood samples were taken from thetail after 4 h and 24 h and incubated on ice. Clotted blood wascentrifuged (10 000 g, 10 min, 4° C.) and serum samples were stored at−80° C. Activity of alanine aminotransaminase was determined by anenzymatic assay (BIOO Scientific, Austin, Tex., USA). To determinecaspase-3 activity in the liver tissue, mice were sacrificed after 24 h(positive control after 5 h) and liver biopsies were taken. Homogenateswere prepared in lysis buffer (200 mM NaCl, 20 mM Tris, 1% NP-40, pH7.4). 10 μg of protein were analyzed by conversion of the fluorogenicsubstrate Ac-DMQD-AMC (Enzo Life Sciences). Caspase activity in PHH andHuh-7 cells was determined as published by Seidel et al. (Hepatology2005, 42:113-120).

Example 3: Xenograft Mouse Tumor Model

8-week-old female NMRI nu/nu mice (Janvier) were injected s.c. with3×10⁶ Colo205 cells in 100 μl PBS at left and right dorsal sides.Treatment started 6 days after tumor cell inoculation when tumorsreached about 100 mm³. Mice received 8 daily i.p. injections of 0.45nmol of the affinity-purified TRAIL fusion proteins according to SEQ IDNOs: 104, 97, and 102, respectively. On day 1, 3, 5 and 7 of treatment,mice received additionally 5 μg bortezomib in 100 μl PBS i.p. threehours before protein injection. The control groups received 100 μl PBSor 5 μg bortezomib at the same time intervals. Tumor growth wasmonitored as described in Schneider et al. (Cell Death Dis. 2010; 1:e68) and Kim et al. (Bioconjug. Chem. 2011; 22: 1631-1637). The Tukey'stest was applied for statistics.

Description of Results Disclosed in Examples 1, 2 and 3

The main aim of the shown examples was the improvement of theproapoptotic activity of scTRAIL fusion proteins under retention oftheir tumor selectivity, i.e. non-reactivity towards normal,non-malignant tissue. In principle, the same rules apply for otherproapoptotic members of the TNF family, as well as all othernon-apoptotic, tissue and immune-regulatory TNF ligands that areinactive as soluble ligands or require membrane targeting to restricttheir activity to the relevant tissue or cell types. The examples arealso not restricted to the specific target antigen used exemplarily(EGFR), but apply, in principle, to all other tissue or cell selectivetargets, including, for tumor therapeutic purposes, tumor stroma markerssuch as fibroblast activation protein.

Construction and Preparation of scTRAIL Fusion Proteins

For generation of functionally improved scFv-TRAIL fusion proteins,first, the genetic code of scTRAIL was adapted for higher protein yieldsin mammalian expression systems. Among the various linker motifssuitable to connect 3 Trail molecules (components A) the (GGGS)1-4motifs were tested, with the shorter linkers (GGGS)₁ (SEQ ID NO: 47) and(GGGS)₂ (SEQ ID NO: 48) being superior to longer linkers with respect toprotein stability, tendency to aggregate and display identical or betterapoptosis inducing activity.

The inventors used EGFR targeting as a model system. Like other membersof the erbB family of receptor tyrosine kinases, the EGFR (erbB1) is anestablished tumor marker, which is overexpressed in several carcinomas,including lung and liver cancer (Olayioye et al, 2000). For generationof the EGFR specific fusion protein of SEQ ID NO: 96, the construct wasN-terminally fused with component B, a humanized and codon-optimizedantibody fragment derived from the anti-EGFR mAb Cetuximab (C225)(Naramura et al, 1993) (SEQ ID NO: 94). A FLAG tag (F) was placedN-terminal of component B for purification and detection purposes (FIG.1A). TRAIL bioactivity depends on the oligomerization state, inparticular relevant for TRAILR2 (DR5), which is poorly activated bysoluble, trimeric forms of TRAIL (Wajant et al, 2001). For SEQ ID NO:102 the linker L between V_(H) and V_(L) was shortened from (GGGGS)₃(SEQ ID NO: 52) to GGGGS (SEQ ID NO: 50). In an independent approach toimprove basal protein stability and protection from proteolyticprocessing during expression culture conditions, two variants of SEQ IDNO: 96 were designed, in which i) the linker X connecting component Aand B comprised two N-glycosylation sites, yielding a monomericglycosylated form (SEQ ID NO:97) and ii) in addition, the two glycinlinkers (P) connecting the three TRAIL components were replaced bylinkers (P1, P2) each comprising two N-glycosylation sites, too (FIG. 1)(SEQ ID NO: 98).

Following expression in stably transfected HEK293 cells, thepurification of SEQ ID NO: 96 and SEQ ID NO: 102 was accomplished bothby IMAC due to intrinsic histidine residues of TRAIL and by M2 mAbaffinity chromatography. For example, yields of >3 mg highly pureprotein per liter cell culture supernatant were achievable for bothfusion proteins. SDS-PAGE and Western blot analysis of the purifiedproteins revealed single protein bands with an approx. molecular mass of70 kDa and 100 kDa for scTRAIL (SEQ ID NO: 104) and EGFR specificV_(H)-V_(L)-scTRAIL fusion proteins SEQ ID NOs: 96 and 102,respectively, matching the expected calculated molecular masses of thesingle stranded monomers of 68, 93 and 94 kDa (FIG. 2). The apparentmolecular mass of SEQ ID NO: 97 was increased compared to SEQ ID NO: 96,in accordance with effective glycosylation of the introduced linker.N-glycosidase treatment of SEQ ID NO: 97 resulted in a protein with amolecular mass essentially identical to that of its non-glycosylatedderivative. The introduction of N-glycosylation sites in SEQ ID NO: 96improved protein stability and protection from degradation during theproduction process, evident from strong reduction of degradationproducts present in culture supernatants (not shown). This allows asingle-step purification of glycosylated variants such as SEQ ID NO: 97with M2 agarose to gain a purification grade comparable to that ofnon-glycosylated fusion proteins after a two-step purification and thusoverall higher yields of purified, bioactive protein.

The gel filtration analysis of fusion proteins (SEQ ID NO: 104) and SEQID NO: 96 (both, with and without glycosylation) indicated that themajority of protein (>94%) exists as a monomer (FIG. 3), whereasretention times decrease from scTRAIL (SEQ ID NO: 104) to SEQ ID NO: 97,according to the increase in molecular size. Concerning scTRAIL (SEQ IDNO: 104), the molecular mass deduced from SEC was slightly lowercompared to that calculated from SDS-PAGE (FIG. 2), which is acharacteristic of this molecule and not a hint for degradation(Schneider et al, 2010). Interestingly, SEC of SEQ ID NO: 102 revealedthe presence of two peaks. The major peak near 200 kDa could beattributed to a dimer, whereas the minor peak could represent trimericand/or tetrameric forms of the fusion protein.

TRAIL fusion proteins comprising an albumin binding domain (ABD) eitherbetween component A and component B (SEQ ID NO: 125) or at theC-terminus of the fusion protein (SEQ ID NO: 126) have been purifiedfrom HEK293 cells as described above. The increase in molecular weight,which is essentially the result of the introduction of the ABD, can beseen on the Coomassie stained SDS-PAGE gel depicted in FIG. 16.

Size exclusion chromatographie experiments showed that the TRAIL fusionproteins comprising an albumin binding domain are indeed capable ofbinding both, humen and mouse serum albumin (FIG. 17).

EGFR-Specific Binding of scTRAIL Fusion Proteins

The specific antigen binding of various scTRAIL fusion proteins toEGFR-positive cells was analysed by flow cytometry of two HCC celllines. Whereas EGFRs in HepG2 cells were barely detectable and thusconsidered target antigen low/negative, EGFR expression was clearlyrevealed in Huh-7 cells (FIG. 4), although EGFR levels in this HCC lineappear moderate compared with EGFR overexpressing A431 cells (data notshown). Consistent with this, the binding of labelled Cetuximab to theEGFR-positive Huh-7 cells can be blocked by preincubation with 0.5 μM ofSEQ ID NO: 102 (FIG. 5). Incubation of HepG2 and Huh-7 cells with SEQ IDNO: 96 or SEQ ID NO: 102 resulted in binding of the proteins to bothcell lines, but competition of fusion protein binding by preincubationof cells with the anti-EGFR huC225 (2 μM) was only possible on Huh-7cells (FIG. 2C). The intermediate fluorescence signal observed uponcompetition of fusion protein binding to Huh-7 cells likely reflectsbinding of the TRAIL domain (component A of the fusion protein) to TRAILreceptors. This is consistent with the weaker and non-blockable bindingof SEQ ID NO: 96 and SEQ ID NO: 102 on EGFR low/neg. HepG2 cells.Binding competition of both fusion proteins to EGFR-positive cells is anindicator for the structural integrity and functionality of thetargeting domain (component B).

Quantitative binding studies of TRAIL fusion proteins to EGFR+, DR4+5+NCI-H460 cells revealed significantly different (P=0.003) EC50 valuesfor TRAIL fusion protein according to SEQ ID NO: 97 (3.6±0.3×10⁻¹⁰ M)and TRAIL fusion protein according to SEQ ID NO: 102 (1.6±0.3×10⁻¹⁰ M),implicating an avidity effect of the specific molecular composition ofthe divalent TRAIL fusion protein according to SEQ ID NO: 102 andtherefore potentially superior targeting compared to TRAIL fusionprotein according to SEQ ID NO: 97 (FIG. 12A). Furthermore, it has beeninvestigated whether the TRAIL fusion protein according to SEQ ID NO:102 exhibits the functional activity of blocking EGF-induced EGFRautophosphorylation. Cetuximab served as a positive control in thisexperiment. Functional blocking of EGF-stimulated receptor activation bythe divalent TRAIL fusion protein according to SEQ ID NO: 102 could bedemonstrated for both Colo205 (FIG. 12B, left panel) and Huh7 (FIG. 12B,right panel) cells.

Target-Independent Induction of Cell Death by scTRAIL Fusion Proteins

To investigate the basic bioactivity of scTRAIL fusion proteins withoutthe influence of targeting domains, we first analysed cell deathinduction on the target-negative cell lines HepG2 (hepatoma) and Jurkat(T cell leukemia) and compared it with a non targeting scTRAIL molecule.On all cell lines analysed, SEQ ID NO: 102 exerted an approximatelytenfold increased bioactivity compared to SEQ ID NO: 96 or itsglycosylated form (SEQ ID NO: 97). As a reference, the bioactivity of acommercially available highly active TRAIL preparation, so-called‘KillerTRAIL’(Enzo Lifesciences), was found to be comparable with theactivity of SEQ ID NO: 96. Due to the low or even deficient targetantigen expression of HepG2 cells, the apoptosis inducing activity ofSEQ ID NO: 102 and SEQ ID NO: 96 was not influenced by the presence ofan at least 7-fold excess (70 nM) of Cetuximab (not shown). Thebioactivity of SEQ ID NO: 96 on target negative cells did not differfrom the one of SEQ ID NO: 104 (scTRAIL). On EGFR-negative,DR4-DR5^(weak) Jurkat cells, which are known to be sensitive forapoptosis induced by TRAIL complexes but not by soluble TRAIL, we founda higher apoptosis-inducing activity of SEQ ID NO: 102 compared toKillerTRAIL and no reactivity towards the SEQ ID NOs: 96 and 104 (FIG.6).

EGFR-Directed Enhancement of Cell Death by scTRAIL Fusion Proteins

The EGFR-positive liver carcinoma cell line Huh-7 was chosen todemonstrate the enhancement in bioactivity achievable due to thereceptor targeting capacity of SEQ ID NO: 96 and SEQ ID NO: 102.Compared to scTRAIL (SEQ ID NO: 104), SEQ ID NO: 96 showed tenfoldbetter apoptosis-inducing activity on Huh-7 cells (FIG. 7). Thecompetition of this bioactivity with an excess of Cetuximab (70 nM)revealed a right shift of EC50 of SEQ ID NO: 96 in the same order ofmagnitude, pointing to the functionality of EGFR targeting responsiblefor the improvement of scTRAIL bioactivity. Glycosylated SEQ ID NO: 97exerted identical bioactivity compared to its non-glycosylated variant,indicating that glycosylation at this site did not impact bioactivity.SEQ ID NO: 102 showed a tenfold enhanced bioactivity in relation to SEQID NO: 96. The competition of activity of SEQ ID NO: 102 with the samemolar excess of Cetuximab also resulted in a comparable shift in thedose response curve, confirming that targeting further improves thealready increased bioactivity of this fusion protein. Furthermore,dimeric TRAIL fusion proteins comprising an albumin binding domain (ABD)(SEQ ID NOs: 125 and 126) exhibited bioactivity comparable to thedimeric TRAIL fusion protein lacking an ABD (SEQ ID NO: 102) indicatingthat the introduction of an ABD as performed for TRAIL fusion proteinsaccording to SEQ ID NOs: 125 and 126 does not significantly influencethe bioactivity of EGFR-directed enhancement of cell death (FIG. 18).

In another experimental setup, Huh-7 cells were pretreated with a fixeddose of the various TRAIL fusion proteins followed by titration of theapoptosis sensitizer Bortezomib (FIG. 8). At a protein concentration of1 nM and above, the tested SEQ ID NOs: 102, 96 and 104 were nearlyequally efficient in induction of complete apoptosis in these cells whensensitized with Bortezomib. In contrast, at protein concentrations of0.1 nM and below, a strong synergistic effect of Bortezomibsensitization and the EGFR targeting ability of the constructs becamevisible. At a protein concentration of 0.05 nM, only SEQ ID NOs: 96 and102 were able to synergize with Bortezomib, whereby SEQ ID NO: 102showed higher bioactivity compared to SEQ ID NO: 96 at thisconcentration. Superior activity of SEQ ID NO: 102 was even moreapparent at 0.01 nM of fusion proteins (FIG. 8).

A nearly complete block of cell death by either pan-caspase (zVADfmk) orcaspase-3 selective (zDEVDfmk) inhibitors (FIG. 13A) and failure ofnecrostatin-1 to prevent or reduce cell death (data not shown) indicatedthat Huh-7 and Colo205 undergo predominantly apoptotic cell death upontreatment with TRAIL fusion proteins. Cetuximab blocked EGF-inducedautophosphorylation of EGF receptors (FIG. 12B). Further, cetuximab byitself, though blocking EGF-induced autophosphorylation of EGFR inColo205 and in Huh7 cells (FIG. 12B), did not substantially affectgrowth of these two cancer cell lines in a 4-day culture (FIG. 13B, C).Likewise, when SEQ ID NO: 102-induced apoptosis was prevented, either bypresence of neutralizing anti-TRAIL antibodies in SEQ ID NO: 102 treatedColo205 cell cultures (FIG. 13B) or by treating Huh-7 cell cultures withpan-caspase inhibitors (FIG. 13C), only a marginal growth inhibition wasnoted during the 4 day observation period. Together, for the cells andthe in vitro conditions studied here, the data indicate that i) SEQ IDNO: 102-induced cell death requires TRAIL signaling and ii) blockingEGFR function by the SEQ ID NO: 102 does not contribute to rapidapoptosis induction.

Binding Affinity of SEQ ID NO: 96 and SEQ ID NO: 102 to Cells

The specific molecular composition of SEQ ID NO: 102 implies avidityeffects and thus potential superior targeting functions as compared toSEQ ID NO: 96. Therefore, we determined dissociation constants of bothfusion proteins on EGFR-positive, DR4+5+ NCI-H460 cells underequilibrium binding conditions at 4° C. by indirect immunofluorescenceflow cytometry with an anti-TRAIL antibody. The K_(D) of the interactionbetween the scTRAIL fusion proteins and NCI-H460 cells at 4° C. wasdetermined by Lineweaver-Burk kinetic analysis (Lineweaver and Burk,1934; Benedict et al, 1997) and was found to be 4-fold lower for thefusion protein of SEQ ID NO: 102 (6.5±0.9×10⁻⁸ M for SEQ ID NO: 96 and1.7±1.2×10⁻⁸ M for SEQ ID NO: 102, respectively). In principle, themeasured K_(D) values reflect cell surface interactions of bothfunctional domains in the fusion protein, the EGFR targeting domain(Component B) and the TRAIL domain, (components A), with theirrespective receptors. However, because SEQ ID NO: 102 binding toTRAILR+, EGFR negative cells such as Jurkat resulted in only very weaksignals in this assay (data not shown), we reason that the signalsrevealed for NCI-H460 are largely due to binding of the fusion proteinvia its V_(H)-V_(L) domain to EGFRs. In fact, under the assay conditions(4° C.) applied, dynamic clustering of TRAILR that could account forstable receptor ligand interactions and thus apparent enhanced affinityis prevented. Therefore, we attribute this increased affinity largely toan avidity effect of the bivalent targeting domain (component B) of thisparticular fusion protein (SEQ ID NO: 102).

Lack of Systemic Toxicity of SEQ ID NO: 102 and Pharmacokinetics

To assess whether the strongly increased bioactivity of SEQ ID NO: 102in vitro diminishes the advantageous tumor selectivity of TRAIL, westudied systemic tolerance and effects of in vivo application on thereportedly most sensitive organ concerning untolerable TRAIL sideeffects, the liver. Groups of 3 CD1 mice were treated intraperitonallywith indicated reagents (FIG. 9): neg. control: PBS; pos. control:aggregated FasL fusion protein; SEQ ID NO: 102 and SEQ ID NO: 96 (A)Plasma samples were prepared after 4 h and 24 h and the activity ofalanine aminotransferase (ALT) was assayed using an enzymatic assay.Dashed line indicates upper normal level of ALT (35-50 U/L). (B) Micewere sacrificed after 24 h except for pos. control (animals treated withan aggregated FasL fusion protein show phenotypic signs of severesystemic toxicity after 2-4 hrs and die after ˜5 hrs, samples were takenafter 4 hrs) and liver biopsies were taken for determination ofcaspase-3 activity using a specific AMC-coupled peptide substrate. Thedata clearly show that the SEQ ID NO: 102, despite its stronglyincreased apoptotic activity in vitro on target positive tumor celllines (compared to non-targeted scTRAIL and the most active commerciallyavailable TRAIL) remains systemically well tolerated at doses up to 3mg/kg in mouse models. Moreover, biochemical parameters of organ (liver)specific pathology confirm the phenotypic tolerance to this TRAIL fusionproteins, with only a transient marginal increase in ALT values to theupper normal limit 4 hrs after application. Lack of caspase activationand baseline ALT after 24 hrs, when bioactive fusion protein is stilldetectable in the blood (Tα_(1/2)=2h, Tβ_(1/2) 3h; plasma conc at t=24h:100 ng/ml with an applied iv dose of 100 μg/animal; proof that SEQ IDNO: 102 maintain tumor selectivity and can be safely applied in vivo.

Furthermore, a comparison of Huh-7 hepatoma cells and primary humanhepatocytes (PHH) for caspase-3 activation by SEQ ID NO: 102 in presenceof bortezomib showed a strong, bortezomib-dependent caspase-3 activationin the tumor cells, whereas normal liver cells were neither affected bythe scTRAIL fusion protein alone nor in combination with bortezomib(FIG. 14A). These results were confirmed by immunoblot analysis ofcleaved caspase-3 in Huh-7 and PHH, with no detectable caspaseactivation in combination treated PHH, whereas robust caspase processingwas detectable in sensitive Huh7 carcinoma cells (FIG. 14B).

Antitumoral Activity of SEQ ID NO: 102 in a Xenograft Tumor Model

Given the in vitro data, showing superior bioactivity of the divalentTRAIL fusion protein according to SEQ ID NO: 102 compared with themonovalent TRAIL fusion protein according to SEQ ID NO: 97 or scTRAILaccording to SEQ ID NO: 104 in particular at low protein concentrations,eight doses of 0.45 nmol protein were injected i.p. in a daily regimenin combination with bortezomib cotreatment every second day. Thesystemic treatment started after establishment of solid, vascularizedtumors and tumor growth was monitored for 22 days. Bortezomib treatmentby itself did not interfere with progressive tumor growth, whereasscTRAIL according to SEQ ID NO: 104 and the monovalent TRAIL fusionprotein according to SEQ ID NO: 97 both delayed tumor growth, but at thelow dosage applied, did not induce regression of tumors. In contrast,upon treatment with the divalent TRAIL fusion protein according to SEQID NO: 102, a strong reduction of tumor size and prolonged survival inall animals, with macroscopically undetectable tumors in 11/12(+bortezomib) and 9/12 (w/o bortezomib) cases was recorded (FIG. 15).Interestingly, under the treatment conditions applied there was only aslight, but statistically not significant benefit of cotreatment withbortezomib, although at termination of treatment the combination grouppresented with slower regrowth of tumors (FIG. 15A).

Introduction of an Albumin Binding Domain Increases the In VivoHalf-Life of TRAIL Fusion Proteins

The pharmacokinetics, in particular, the in vivo half-lives for thedimeric TRAIL fusion protein according to SEQ ID NO: 102 lacking analbumin binding domain (ABD) and the dimeric fusion protein comprisingan ABD between component A and component B of the TRAIL fusion protein(SEQ ID NO: 125) have been compared. To this end, 25 μg of fusionproteins were injected i.v. in CD1 mice and serum samples were analyzedat certain time points after injection by ELISA assay (FIG. 19). It hasbeen demonstrated that the in vivo serum half-life increases from about3 hours for the construct without ABD (SEQ ID NO: 102) to about 20 hoursfor the construct comprising an ABD (SEQ ID NO: 125) (FIG. 19). Asindicated above, the constructs comprising an ABD exert a similarbioactivity compared to the constructs lacking an ABD (Figure. 18)indicating that the ABD does not negatively influence bioactivity, butexerts advantageous properties regarding pharmacokinetic properties,such as in vivo serum half-life. Thus, the TRAIL fusion proteins,preferably dimeric TRAIL fusion proteins, as described above comprisingan ABD, such as the dimeric TRAIL fusion proteins according to SEQ IDNO: 125 and 126, are particularly preferred embodiments of thepolypeptide according to the present invention.

The inventors of the present invention have thus provided evidence foran improved concept in targeted cancer therapy. It may be thatpolypeptides according to the present invention form oligomers whilepolypeptides such as SEQ ID NO: 96 remain strictly monomeric. If so, itis surprising that the potential oligomeric structure of thepolypeptides according to the present invention does not result in anincreased systemic toxicity. The presence of higher-order aggregates inpreparations of recombinant TRAIL constructs (e.g. His-TRAIL,crosslinked FLAG-TRAIL) has been reported previously to be responsiblefor an increased toxicity towards some non-malignant tissue cells(reviewed by Koschny et al, 2007). Thus, the inventors provide newformats of highly active and tumor selective TRAIL molecules withimproved in vivo stability and pharmacokinetic properties, thus reachingan unprecedented potential as tumor therapeutic.

The invention claimed is:
 1. A polypeptide comprising: a) at least threecomponents A, each of which comprises the sequence of a TNF homologydomain (THD) of 4-1BBL having at least 90% sequence identity to SEQ IDNO: 31, and b) at least one component B consisting of a V_(L) region anda V_(H) region linked directly to each other with a linker sequence Lwhich has a length of ≤12 amino acids, wherein the V_(L) and V_(H)regions are of an antibody that binds to a cell surface moleculeselected from the group consisting of: a cytokine receptor, a growthfactor receptor, an integrin, a cell adhesion molecule and/or a celltype- or tissue-specific cell surface antigen, cell surface expressedtumor-associated antigens (TAA), and carbohydrates.
 2. The polypeptideaccording to claim 1, wherein the at least three components A areidentical.
 3. The polypeptide according to claim 1, wherein component Acomprises a sequence having at least 95% sequence identity to SEQ IDNOs:
 31. 4. The polypeptide according claim 1, wherein the at least 3components A are directly linked to each other via at least twointervening peptide linkers (peptide linker P).
 5. The polypeptideaccording to claim 4, wherein the at least two peptide linkers P areselected from the group consisting of SEQ ID NOs: 39-90, and
 91. 6. Thepolypeptide according to claim 5, wherein the at least two peptidelinkers P are selected from SEQ ID NOs: 48, 88, and
 90. 7. Thepolypeptide according to claim 1, wherein the V_(L) region is selectedfrom the group consisting of SEQ ID NOs: 92 and 130-132, and wherein theV_(H) region is selected from the group consisting of SEQ ID NOs: 93 and133-135.
 8. The polypeptide according to claim 7, wherein the V_(L)region is SEQ ID NO: 92 and the V_(H) region is SEQ ID NO:
 93. 9. Thepolypeptide according to claim 1, wherein the V_(L) and V_(H) region ofa component B are linked with a linker sequence L selected from thegroup consisting of SEQ ID NOs: 39-51 and 53-78.
 10. The polypeptideaccording to claim 9, wherein the linker sequence L is SEQ ID NO: 50.11. The polypeptide according to claim 1, wherein component B has thesequence of SEQ ID NO: 94, 136, 137 or
 138. 12. The polypeptideaccording to claim 1, wherein the polypeptide comprises a glycosylationmotif and/or is glycosylated and/or comprises an albumin binding domain(ABD).
 13. The polypeptide according to claim 1, wherein the polypeptidecomprises an albumin binding domain (ABD) located between component Aand component B or downstream of component A.
 14. The polypeptideaccording to claim 13, wherein the polypeptide comprises an albuminbinding domain (ABD) located at the C-terminal end of the polypeptide.15. A nucleic acid encoding for the polypeptides according to claim 1.16. The polypeptide complex comprising the polypeptide according toclaim
 1. 17. The polypeptide complex according to claim 16, which is adimeric and/or trimeric complex.
 18. A pharmaceutical compositioncomprising at least one polypeptide according to claim 1, optionallyfurther comprising at least one pharmaceutically acceptable carrier,adjuvant, and/or vehicle.